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1.
J Immunol ; 208(4): 929-940, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35091434

RESUMO

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses, therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.


Assuntos
Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Microfluídica , Neoplasias/imunologia , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Técnicas de Cultura de Células , Feminino , Humanos , Imunização , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Knockout , Microfluídica/métodos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
2.
J Transl Med ; 21(1): 757, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37884933

RESUMO

Liver steatosis, inflammation, and variable degrees of fibrosis are the pathological manifestations of nonalcoholic steatohepatitis (NASH), an aggressive presentation of the most prevalent chronic liver disease in the Western world known as nonalcoholic fatty liver (NAFL). Mitochondrial hepatocyte dysfunction is a primary event that triggers inflammation, affecting Kupffer and hepatic stellate cell behaviour. Here, we consider the role of impaired mitochondrial function caused by lipotoxicity during oxidative stress in hepatocytes. Dysfunction in oxidative phosphorylation and mitochondrial ROS production cause the release of damage-associated molecular patterns from dying hepatocytes, leading to activation of innate immunity and trans-differentiation of hepatic stellate cells, thereby driving fibrosis in NASH.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/patologia , Fígado/patologia , Hepatócitos/patologia , Inflamação/patologia , Fibrose , Mitocôndrias/patologia
3.
J Transl Med ; 21(1): 425, 2023 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-37386590
4.
Antimicrob Agents Chemother ; 59(7): 3808-15, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25870055

RESUMO

Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Esteroides/farmacologia , Bacteriófago Pf1/patogenicidade , Biofilmes/efeitos dos fármacos , Fibrose Cística/microbiologia , Humanos , Lipídeos/farmacologia , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Modelos Biológicos , Pregnanos/farmacologia , Propilaminas/farmacologia , Pseudomonas aeruginosa/patogenicidade , Catelicidinas
5.
Stem Cells ; 32(5): 1337-46, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24446170

RESUMO

MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by four-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235(+)CD41(-)), megakaryocyte (CD41(+)CD42(+)), and myeloid (CD18(+)CD235(-)) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a sixfold enhancement in megakaryocyte production. miR-513a, miR-571, and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34(+) cells derived from peripheral blood. In addition, adult CD34(+) cells express endogenous miR-105 during megakaryocyte differentiation. siRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3'UTR construct and Western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , MicroRNAs/genética , Trombopoese/genética , Regiões 3' não Traduzidas/genética , Adulto , Antígenos CD34/metabolismo , Western Blotting , Antígenos CD18/metabolismo , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Células Eritroides/citologia , Células Eritroides/metabolismo , Citometria de Fluxo , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , MicroRNAs/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Bioorg Med Chem Lett ; 25(14): 2837-43, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26004577

RESUMO

Certain membrane-active cationic steroids are known to also possess both anti-inflammatory and antimicrobial properties. This combined functionality is particularly relevant for potential therapies of infections associated with elevated tissue damage, for example, cystic fibrosis airway disease, a condition characterized by chronic bacterial infections and ongoing inflammation. In this study, six novel cationic glucocorticoids were synthesized using beclomethasone, budesonide, and flumethasone. Products were either monosubstituted or disubstituted, containing one or two steroidal groups, respectively. In vitro evaluation of biological activities demonstrated dual anti-inflammatory and antimicrobial properties with limited cytotoxicity for all synthesized compounds. Budesonide-derived compounds showed the highest degree of both glucocorticoid and antimicrobial properties within their respective mono- and disubstituted categories. Structure-activity analyses revealed that activity was generally related to the potency of the parent glucocorticoid. Taken together, these data indicate that these types of dual acting cationic lipids can be synthesized with the appropriate starting steroid to tailor activities as desired.


Assuntos
Anti-Infecciosos/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/síntese química , Lipídeos/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Cátions/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Escherichia coli/efeitos dos fármacos , Glucocorticoides/síntese química , Glucocorticoides/química , Hemólise/efeitos dos fármacos , Humanos , Lipídeos/síntese química , Lipídeos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Estrutura-Atividade
7.
Front Immunol ; 13: 1015585, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36263022

RESUMO

Activation of T cell responses is essential for effective tumor clearance; however, inducing targeted, potent antigen presentation to stimulate T cell responses remains challenging. We generated Activating Antigen Carriers (AACs) by engineering red blood cells (RBCs) to encapsulate relevant tumor antigens and the adjuvant polyinosinic-polycytidylic acid (poly I:C), for use as a tumor-specific cancer vaccine. The processing method and conditions used to create the AACs promote phosphatidylserine exposure on RBCs and thus harness the natural process of aged RBC clearance to enable targeting of the AACs to endogenous professional antigen presenting cells (APCs) without the use of chemicals or viral vectors. AAC uptake, antigen processing, and presentation by APCs drive antigen-specific activation of T cells, both in mouse in vivo and human in vitro systems, promoting polyfunctionality of CD8+ T cells and, in a tumor model, driving high levels of antigen-specific CD8+ T cell infiltration and tumor killing. The efficacy of AAC therapy was further enhanced by combination with the chemotherapeutic agent Cisplatin. In summary, these findings support AACs as a potential vector-free immunotherapy strategy to enable potent antigen presentation and T cell stimulation by endogenous APCs with broad therapeutic potential.


Assuntos
Vacinas Anticâncer , Camundongos , Humanos , Animais , Idoso , Poli I-C , Fosfatidilserinas , Cisplatino , Antígenos de Neoplasias , Eritrócitos
8.
J Ind Microbiol Biotechnol ; 38(11): 1809-20, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21487833

RESUMO

Natural products have long served as rich sources of drugs possessing a wide range of pharmacological activities. The discovery and development of natural product drug candidates is often hampered by the inability to efficiently scale and produce a molecule of interest, due to inherent qualities of the native producer. Heterologous biosynthesis in an engineering and process-friendly host emerged as an option to produce complex natural products. Escherichia coli has previously been utilized to produce complex precursors to two popular natural product drugs, erythromycin and paclitaxel. These two molecules represent two of the largest classes of natural products, polyketides and isoprenoids, respectively. In this study, we have developed a platform E. coli strain capable of simultaneous production of both product precursors at titers greater than 15 mg l(-1). The utilization of a two-phase batch bioreactor allowed for very strong in situ separation (having a partitioning coefficient of greater than 5,000), which would facilitate downstream purification processes. The system developed here could also be used in metagenomic studies to screen environmental DNA for natural product discovery and preliminary production experiments.


Assuntos
Alcenos/metabolismo , Diterpenos/metabolismo , Eritromicina/análogos & derivados , Escherichia coli/metabolismo , Alcenos/química , Produtos Biológicos/metabolismo , Reatores Biológicos , Diterpenos/química , Eritromicina/biossíntese , Eritromicina/química , Policetídeos/metabolismo , Terpenos/metabolismo
9.
Hum Gene Ther ; 25(11): 966-76, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25144316

RESUMO

Patients with cystic fibrosis (CF) often suffer chronic lung infection with concomitant inflammation, a setting that may reduce the efficacy of gene transfer. While gene therapy development for CF often involves viral-based vectors, little is known about gene transfer in the context of an infected airway. In this study, three mouse models were established to evaluate adeno-associated virus (AAV) gene transfer in such an environment. Bordetella bronchiseptica RB50 was used in a chronic, nonlethal respiratory infection in C57BL/6 mice. An inoculum of ∼10(5) CFU allowed B. bronchiseptica RB50 to persist in the upper and lower respiratory tracts for at least 21 days. In this infection model, administration of an AAV vector on day 2 resulted in 2.8-fold reduction of reporter gene expression compared with that observed in uninfected controls. Postponement of AAV administration to day 14 resulted in an even greater (eightfold) reduction of reporter gene expression, when compared with uninfected controls. In another infection model, Pseudomonas aeruginosa PAO1 was used to infect surfactant protein D (SP-D) or surfactant protein A (SP-A) knockout (KO) mice. With an inoculum of ∼10(5) CFU, infection persisted for 2 days in the nasal cavity of either mouse model. Reporter gene expression was approximately ∼2.5-fold lower compared with uninfected mice. In the SP-D KO model, postponement of AAV administration to day 9 postinfection resulted in only a two fold reduction in reporter gene expression, when compared with expression seen in uninfected controls. These results confirm that respiratory infections, both ongoing and recently resolved, decrease the efficacy of AAV-mediated gene transfer.


Assuntos
Fibrose Cística/terapia , Dependovirus/genética , Transdução Genética , Doença Aguda , Animais , Infecções por Bordetella/microbiologia , Doença Crônica , Fibrose Cística/complicações , Humanos , Masculino , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/microbiologia , Infecções Respiratórias/microbiologia
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