ObjectivesOpen-labelled clinical trials suggested that low-dose IL-2 might be effective in treatment of systemic lupus erythematosus (SLE). A double-blind and placebo-controlled trial is required to formally evaluate the safety and efficacy of low-dose IL-2 therapy.MethodsA randomised, double-blind and placebo-controlled clinical trial was designed to treat 60 patients with active SLE. These patients received either IL-2 (n=30) or placebo (n=30) with standard treatment for 12 weeks, and were followed up for additional 12 weeks. IL-2 at a dose of 1 million IU or placebo was administered subcutaneously every other day for 2 weeks and followed by a 2-week break as one treatment cycle. The primary endpoint was the SLE Responder Index-4 (SRI-4) at week 12. The secondary endpoints were other clinical responses, safety and dynamics of immune cell subsets.ResultsAt week 12, the SRI-4 response rates were 55.17% and 30.00% for IL-2 and placebo, respectively (p=0.052). At week 24, the SRI-4 response rate of IL-2 group was 65.52%, compared with 36.67% of the placebo group (p=0.027). The primary endpoint was not met at week 12. Low-dose IL-2 treatment resulted in 53.85% (7/13) complete remission in patients with lupus nephritis, compared with 16.67% (2/12) in the placebo group (p=0.036). No serious infection was observed in the IL-2 group, but two in placebo group. Besides expansion of regulatory T cells, low-dose IL-2 may also sustain cellular immunity with enhanced natural killer cells.ConclusionsLow-dose IL-2 might be effective and tolerated in treatment of SLE.Trial registration numberClinicalTrials.gov Registries (NCT02465580 and NCT02932137).

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.

Sonoporation has been exploited as a promising strategy for intracellular drug and gene delivery. The technique uses ultrasound to generate pores on the cell membrane to allow entry of extracellular agents into the cell. Resealing of these non-specific pores is a key factor determining both the uptake and post-ultrasound cell survival. This study examined the effects of extracellular Ca 2+ on membrane resealing in sonoporation, using Xenopus oocytes as a model system. The cells were exposed to tone burst ultrasound (1.06 MHz, duration 0.2 s, acoustic pressure 0.3 MPa) in the presence of 0.1% Definity ® at various extracellular [Ca 2+ ] (0-3 mM). Sonoporation inception and resealing in a single cell were monitored in real time via the transmembrane current of the cell under voltage clamp. The time-resolved measurements of transmembrane current revealed the involvement of two or more Ca 2+ related processes with different rate constants and characteristics. Rapid resealing occurred immediately after ultrasound application followed by a much slower resealing process. Complete resealing required [Ca 2+ ] above 0.54 mM. The cells resealed in 6-26 s at 1.8 mM Ca 2+ , but took longer at lower concentrations, up to 58 ~170 s at 0.54 mM Ca 2+ .

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actinenriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1 ؊/؊ mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.

Sonoporation uses ultrasound (US) to generate transient non-selective pores on the cell membrane and has been exploited as a non-viral intracellular drug and gene delivery strategy. The pore size determines the size of agents that can be delivered into the cytoplasm using the technique. However, measurements of the dynamic, submicron-scale pores have not been readily available. Electron microscopy or atomic force microscopy has been used to gauge pore size but such techniques are intrinsically limited to post US measurements that may not accurately reveal the relevant information. As previously demonstrated, changes of the transmembrane current (TMC) of a single cell under voltage clamp can be used for monitoring sonoporation in real time. Because the TMC is related to the diffusion of ions through the pores on the membrane, it can potentially provide information of the pore size generated in sonoporation. Using Xenopus laevis oocytes as the model system, the TMC of single cells under voltage clamp was measured in real time to assess formation of pores on the membrane in sonoporation. The cells were exposed to US (0.2 s, 0.3 MPa, 1.075 MHz) in the presence of Definity™ microbubbles. Experiments were designed to obtain the TMC corresponding to a single pore on the membrane. The size of the pores was estimated from an electro-diffusion model that relates the TMC with pore size from the ion transport through the pores on the membrane. The mean radius of single pores was determined to be 110 nm with standard deviation of 40 nm. This study reports the first results of pore size from the TMC measured using the voltage clamp technique.

Effective treatment of solid tumors requires homogenous distribution of anticancer drugs within the entire tumor volume to deliver lethal concentrations to resistant cancer cells and tumorinitiating cancer stem cells. However, penetration of small molecular weight chemotherapeutic agents and drug-loaded polymeric and lipid particles into the hypoxic and necrotic regions of solid tumors remains a significant challenge. This article reports the results of pulsed ultrasound enhanced penetration of nano-sized fluorescent particles into MCF-7 breast cancer spheroids (300-350 μm diameter) as a function of particle size and charge. With pulsed ultrasound application in the presence of microbubbles, small (20 nm) particles achieve 6-20 folds higher penetration and concentration in the spheroid's core compared to those not exposed to ultrasound. Increase in particle size to 40 nm and 100 nm results in their effective penetration into the spheroid's core to 9 and 3 folds, respectively. In addition, anionic carboxylate particles achieved higher penetration (2.3, 3.7, and 4.7 folds) into the core (0.25r) of MCF-7 breast cancer spheroids compared to neutral (2.2, 1.9, and 2.4 folds) and cationic particles (1.5, 1.4 and 1.9 folds) upon US exposure for 30, 60, and 90 seconds under the same experimental conditions. These results demonstrate the feasibility of utilizing pulsed ultrasound to increase the penetration of nano-sized particles into MCF-7 spheroids mimicking tumor tissue. The effects of particle properties on the penetration enhancement were also illustrated.

RNA editing within the pore loop controls the pharmacology and permeation properties of ion channels formed by neuronal AMPA and kainate receptor subunits. Genomic sequences for the glutamate receptor 2 (GluR2) subunit of AMPA receptors and the GluR5 and GluR6 subunits of kainate receptors all encode a neutral glutamine (Q) residue within the channel pore that can be converted by RNA editing to a positively charged arginine (R). Receptors comprised of unedited subunits are permeable to calcium and display inwardly rectifying current-voltage relationships, because of blocking of outward current by intracellular polyamines. In contrast, receptors that include edited subunits conduct less calcium, resist polyamine block, and have relatively linear current-voltage relationships. We showed previously that cis-unsaturated fatty acids, including arachidonic acid and docosahexanoic acid, exert a potent block of native kainate receptors as well as homomeric recombinant receptors formed by transfection of heterologous cells with cDNA for the GluR6(R) subunit. Here, we show that fatty acid blockade of recombinant homomeric and heteromeric kainate receptors is strongly dependent on editing at the Q/R site. Recombinant channels that include unedited subunits exhibit significantly weaker block than channels made up of fully edited subunits. Inhibition of fully edited channels is equivalent at voltages from Ϫ70 to ϩ40 mV and is noncompetitive, consistent with allosteric regulation of channel function.