Non-syncytium-inducing (NSI) strains of HIV-1 prevail among most infected children, including pediatric patients who develop advanced disease, severe immune suppression, and die. A study was designed to address the hypothesis that genotypic and/or phenotypic markers can distinguish NSI viruses isolated during early infection from NSI viruses found in advanced disease. Primary HIV-1 isolates, which were obtained from 43 children, adolescents, and adults who displayed a cross-section of clinical disease and immune suppression but were untreated by protease inhibitor antiretroviral therapy, were characterized for replication phenotype in different cell types. Most individuals (81%) harbored NSI viruses and almost half had progressed to advanced disease or severe immune deficiency. About 51% of NSI isolates produced low levels of p24 antigen (median, 142 pg/ml) in monocyte-derived macrophages (MDMs), 31% produced medium levels (median, 1584 pg/ml), and 17% produced high levels (median, 81,548 pg/ml) (p < 0.001). Seven of eight syncytium-inducing isolates also replicated in MDMs and displayed a dual-tropic phenotype that was associated with advanced disease. Replication of NSI viruses in MDMs varied as much as 100- to 1000-fold and was independent of replication in peripheral blood mononuclear cells. Replication in MDMs provided a clear biological feature to distinguish among viruses that were otherwise identical by NSI phenotype, V3 genotype, and CCR5 coreceptor usage. Low-level MDM replication was characteristic of viruses isolated from asymptomatic individuals, including long-term survivors. Enhanced MDM replication was related to morbidity and mortality among patients. Replication levels in MDMs provide a novel prognostic indicator of pathogenic potential by NSI viruses.
ABSTRACT Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre‐miR‐199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV‐1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre‐miR‐199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre‐miR‐199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre‐miR‐199a. The TAT‐TAR interaction enhanced the loading of the miR‐199a into EVs by 65‐fold. Endogenously loaded EVs were ineffective at delivering active miR‐199a‐3p therapeutic to recipient SK‐Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.
Abstract : War is our business. To do it well, our graduates must have a firm understanding of military power and its place within the American Republic. Since the 1954 founding of the United States Air Force Academy (USAFA), the study of the context, theory, and application of military power has remained central to the institution's mission. Indeed, one may argue such expertise is the essence of a military academy education. Nonetheless, throughout the Academy's existence, arguments have raged over the disciplinary content and organizational ownership of this essence. Just as the founders, leaders, and scholars of the Academy sought to blend officer education and training, the earliest stirrings of the Department of Military and Strategic Studies tried to reconcile the theoretical aspects of military power with practical applications. Today, this Department is one of only a handful at North American undergraduate or graduate institutions offering an accredited disciplinary major focused on understanding the theory and application of military power regardless of time and context. Given the importance of military power to American security, the broader scholarly enterprise warrants castigation for its neglect.
ABSTRACT Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV‐induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR‐199a‐3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.
