Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ-on-a-Chip.

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales.

Hierarchical assembly of nanostructured coating for siRNA-based dual therapy of bone regeneration and revascularization.

Advancing bone implant engineering offers the opportunity to overcome crucial medical challenges and improve clinical outcomes. Although the establishment of a functional vascular network is crucial for bone development, its regeneration inside bone tissue has only received limited attention to date. Herein, we utilize siRNA-decorated particles to engineer a hierarchical nanostructured coating on clinically used titanium implants for the synergistic regeneration of skeletal and vascular tissues. Specifically, an siRNA was designed to target the regulation of cathepsin K and conjugated on nanoparticles. The functionalized nanoparticles were assembled onto the bone implant to form a hierarchical nanostructured coating. By regulating mRNA transcription, the coating significantly promotes cell viability and growth factor release related to vascularization. Moreover, microchip-based experiments demonstrate that the nanostructured coating facilitates macrophage-induced synergy in up-regulation of at least seven bone and vascular growth factors. Ovariectomized rat and comprehensive beagle dog models highlight that this siRNA-integrated nanostructured coating possesses all the key traits of a clinically promising candidate to address the myriad of challenges associated with bone regeneration.

A rapid triage test for active pulmonary tuberculosis in adult patients with persistent cough.

Improved tuberculosis (TB) prevention and control depend critically on the development of a simple, readily accessible rapid triage test to stratify TB risk. We hypothesized that a blood protein-based host response signature for active TB (ATB) could distinguish it from other TB-like disease (OTD) in adult patients with persistent cough, thereby providing a foundation for a point-of-care (POC) triage test for ATB. Three adult cohorts consisting of ATB suspects were recruited. A bead-based immunoassay and machine learning algorithms identified a panel of four host blood proteins, interleukin-6 (IL-6), IL-8, IL-18, and vascular endothelial growth factor (VEGF), that distinguished ATB from OTD. An ultrasensitive POC-amenable single-molecule array (Simoa) panel was configured, and the ATB diagnostic algorithm underwent blind validation in an independent, multinational cohort in which ATB was distinguished from OTD with receiver operator characteristic-area under the curve (ROC-AUC) of 0.80 [95% confidence interval (CI), 0.75 to 0.85], 80% sensitivity (95% CI, 73 to 85%), and 65% specificity (95% CI, 57 to 71%). When host antibodies against TB antigen Ag85B were added to the panel, performance improved to 86% sensitivity and 69% specificity. A blood-based host response panel consisting of four proteins and antibodies to one TB antigen can help to differentiate ATB from other causes of persistent cough in patients with and without HIV infection from Africa, Asia, and South America. Performance characteristics approach World Health Organization (WHO) target product profile accuracy requirements and may provide the foundation for an urgently needed blood-based POC TB triage test.

Single Molecule Arrays for ultra-sensitive detection of rat cytokines in serum.

Rats are used as animal models for many human diseases. Cytokines can serve as biomarkers indicative of these diseases or disease states. Techniques for measuring cytokine expression levels often do not provide the sensitivity needed to measure these biomarkers in biological fluids because the concentrations of many cytokines are below the detection limits of conventional methods. In this paper, we present ultra-sensitive digital immunoassays using Single Molecule Arrays (Simoa) for seven rat cytokines: TNF-α, IL-10, IL-17F, GM-CSF, IFN-γ, IL-4, and IL-1α. These ultra-sensitive immunoassays have limits of detection (LODs) in the femtomolar range and provide the ability to measure rat cytokines in serum below the LODs of conventional immunoassays. We also measured these cytokines in healthy rat serum to obtain baseline levels. The ability to measure cytokines present at low concentrations in rat serum will facilitate future studies of disease using rats as animal models.

Direct fluorescent measurement of blood potassium with polymeric optical sensors based on upconverting nanomaterials.

We proposed here a potassium selective optode incorporating NaYF4:Er,Yb upconverting nanorods and chromoionophore ETH 5294 together in hydrophobic polymer matrixes and the response of the optode is based on changes in the upconverting luminescence intensity induced from the absorption change of the proton sensitive chromoionophore. The optode was used for determination of the potassium content in the whole blood sample for the first time. Because the excitation source of 980 nm, as well as the emission wavelength, lies in the near-infrared region, the background absorption and autofluorescence of the biological sample could be eliminated, and ensure the sensitivity and selectivity of the sensor. The potassium levels of sheep plasma and whole blood samples obtained by the optode were comparable with the results obtained by ICPMS and ISE methods, providing possibilities for further application in clinical diagnosis.

Polymeric optodes based on upconverting nanorods for fluorescent measurements of pH and metal ions in blood samples.

Optical thin films incorporating NaYF(4):Er,Yb upconverting nanorods and chromoionophore ETH 5418 in hydrophobic polymer matrixes have been developed for the first time to measure pH and metal ions based on the ion-exchange mechanism. The absorption spectra of protonated and unprotonated ETH 5418 overlap the two emission peaks of upconverting material, respectively, which makes the inert nanorods ion-sensitive. Optodes for pH and metal ions (Na(+), K(+), Ca(2+), and Cu(2+)) were investigated and exhibited excellent sensitivity, selectivity, and reproducibility. Because of excitation by the 980 nm laser source, detection in the near-infrared region at 656 nm, and high quantum yield of the nanorods in hydrophobic membrane, the proposed sensors have been successfully used in whole blood measurements with minimized background absorption and sample autofluorescence.