Droplet microfluidics for functional temporal analysis and cell recovery on demand using microvalves: application in immunotherapies for cancer.

Most common methods of cellular analysis employ the top-down approach (investigating proteomics or genomics directly), thereby destroying the cell, which does not allow the possibility of using the same cell to correlate genomics with functional assays. Herein we describe an approach for single-cell tools that serve as a bottom-up approach. Our technology allows functional phenotyping to be conducted by observing the cytotoxicity of cells and then probe the underlying biology. We have developed a droplet microfluidic device capable of trapping droplets in the array and releasing the droplet of interest selectively using microvalves. Each droplet in the array encapsulates natural killer cells (NK cells) and tumour cells for real-time monitoring of burst kinetics and spatial coordination during killing by single NK cells. Finally, we use the microvalve actuation to selectively release droplets with the desired functional phenotype such as for fast and serial killing of target tumour cells by NK cells. From this perspective, our device allows for investigating first interactions and real-time monitoring of kinetics and later cell recovery on demand for single-cell omic analysis such as single-cell RNA sequencing (scRNA), which to date, is primarily based on in-depth analyses of the entire transcriptome of a relatively low number of cells.

Efficient Production of Murine Uterine Damage Model.

BACKGROUND: Thin or damaged endometrium causes uterine factor-derived infertility resulting in a failure of embryonic implantation. Regeneration of endometrium is a major issue in gynecology and reproductive medicine. Various types of cells and scaffolds were studied to establish an effective therapeutic strategy. For this type of investigations, production of optimal animal models is indispensable. In this study, we tried to establish various murine uterine damage models and compared their features. METHODS: Three to ten-week-old C57BL/6 female mice were anesthetized using isoflurane. Chemical and mechanical methods using ethanol (EtOH) at 70 or 100% and copper scraper were compared to determine the most efficient condition. Damage of uterine tissue was induced either by vaginal or dorsal surgical approach. After 7-10 days, gross and microscopic morphology, safety and efficiency were compared among the groups. RESULTS: Both chemical and mechanical methods resulted in thinner endometrium and reduced number of glands. Gross morphology assessment revealed that the damaged regions of uteri showed various shapes including shrinkage or cystic dilatation of uterine horns. The duration of anesthesia significantly affected recovery after procedure. Uterine damage was most effectively induced by dorsal approach using 100% EtOH treatment compared to mechanical methods. CONCLUSION: Taken together, murine uterine damage models were most successfully established by chemical treatment. This production protocols could be applied further to larger animals such as non-human primate.

Anti-melanogenesis effect of dehydroglyasperin C through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16F1 melanoma cells.

BACKGROUND: In mammals, UV radiation induces melanin synthesis in melanocyte for protecting their skin through the stimulation of α-melanocyte stimulating hormone (α-MSH) from keratinocytes. In this study, the inhibitory effects of dehydroglyasperin C (DGC), an useful component of Glycyrrhiza uralensis (G. uralensis), was investigated on melanogenesis induced by α-melanocyte stimulating hormone (α-MSH) and its mechanisms. METHODS: Melanogenesis suppression effect of DGC on α-MSH induced B16F1 melanoma cells. The cell viability was measured by MTT assay. Expression and phosphorylation of melanogeic protein were conducted using western blot. cAMP acceleration was measured by cAMP immunoassay kit. To investigate whitening mechanism, we used ERK inhibitor (PD98059). RESULTS: DGC decreased intra cellular tyrosinase (TYR) activity and expression of melanin synthesis related proteins (TYR and TRP-1) in a dose-dependent manner on α-MSH induced melanogenesis. In addition, DGC induced the downregulation of MITF (melanocyte-specific transcription factor) through suppression of cAMP-CREB pathway. Also, phosphorylation of extracellular signal regulated kinase (ERK) decreased MITF by DGC treatment. CONCLUSION: Therefore, DGC could be used as a whitening ingredient in skin and clinical usage against hyperpigmentation.

Development of a highly specific and sensitive cadmium and lead microbial biosensor using synthetic CadC-T7 genetic circuitry.

Multiple copies of a cadC homolog encoding a heavy metal-responsive transcription factor were found in the genome of a bacterium isolated from ocean sediment, and the heavy metal responses of the encoded proteins were characterized using a fluorescence reporter assay. Each CadC regulator exhibited distinct specificity in response to heavy metal ions, indicating their potential use as modular heavy metal biosensors. Next, we constructed CadC-controlled T7 RNA transcription systems for intracellular signal amplification, i.e., higher sensitivity. Flow cytometry revealed that cadmium and lead ions could be recognized specifically by CadC-T7 biosensors, which could be combined with a microfluidic platform to generate heavy metal biosensor devices with increased sensitivity. Our results demonstrate the successful development of synthetic CadC-T7 genetic circuitry for use in improved heavy metal biosensor microfluidic devices.

Comparison of intravitreal bevacizumab alone or combined with triamcinolone versus triamcinolone in diabetic macular edema: a randomized clinical trial.

PURPOSE: To compare the effect of an intravitreal injection of bevacizumab alone (IVB) or combined with triamcinolone (IVB/IVT) versus triamcinolone (IVT) in patients with diabetic macular edema (DME). METHODS: In this randomized three-arm clinical trial, eligible eyes were assigned randomly to one of the three study arms: the IVB group, 2 injections of 1.25 mg of bevacizumab with 6-week intervals; the IVB/IVT group, 1.25 mg of IVB with 2 mg of IVT, and the IVT group, 2 mg of IVT. The clinical course of best-corrected visual acuity and central macular thickness by optical coherence tomography was monitored for up to 12 months after the initial injection. RESULTS: One hundred eleven eyes of 105 patients with DME completed 12 months of follow-up. The IVB/IVT group and the IVT group showed better visual acuity and reduced central macular thickness at 6 weeks and 3 months, compared with the IVB group (p = 0.041, p = 0.02 at 6 weeks; p = 0.045, p = 0.043 at 3 months, respectively). However, no significant difference in visual acuity and central macular thickness was observed between the three groups at 12 months (p = 0.088, p = 0.132, respectively). The frequency of retreatment was lower in the IVB/IVT and IVT groups during the 12-month period (p < 0.001). No significant differences in visual acuity or central macular thickness were observed between the IVB/IVT and IVT groups during the follow-up. CONCLUSION: IVB/IVT and IVT showed more pronounced effects during the earlier postinjection period. However, levels of visual acuity or central macular thickness at 12 months were comparable in the three study groups. No beneficial effect of the combination injection was observed.

Surgical outcomes for primary rhegmatogenous retinal detachments in patients with pseudophakia after phacoemulsification.

PURPOSE: To evaluate the clinical features and surgical outcomes for primary rhegmatogenous retinal detachments (RDs) in patients with pseudophakia after phacoemulsification. METHODS: The medical records of patients with pseudophakia after phacoemulsification and intraocular lens implantation who had undergone surgery for primary rhegmatogenous RDs with a minimum duration of follow-up of 12 months were reviewed retrospectively. RESULTS: A total of 104 patients were enrolled in this study and 106 eyes were analyzed. Post-operative retinal attachment was achieved in 87 of the eyes (82.1%) and the final visual acuities (logarithm of the minimum angle of resolution) were improved to 0.65 ± 0.49 from the baseline measurement of 1.51 ± 1.14 (p < 0.001). Re-operations were performed in 24 of the eyes (22.6%) and there were no visible retinal breaks in 30 of the eyes (28.3%). The failure to identify a retinal break during surgery was associated with a lower rate of retinal reattachment, worse final visual acuity, and a higher rate of re-operation (p = 0.002, p = 0.02, and p = 0.002, respectively). The location of the identified retinal break was more common in the superotemporal quadrant than in the other quadrants. CONCLUSIONS: The inability to identify a retinal break during surgery was associated with a poor final outcome. Other factors were less important for the functional and anatomic success in patients with pseudophakic RDs.

Intravitreal bevacizumab (avastin) as an adjuvant for the treatment of posterior scleritis.

We report a case of posterior scleritis effectively managed with intravitreal bevacizumab. A 71-year-old woman was diagnosed with posterior scleritis. Although she was initially treated with systemic steroids, her clinical presentation deteriorated. She was then treated with a single intravitreal injection of bevacizumab and aqueous humor collection. The aqueous level of vascular endothelial growth factor prior to the intravitreal injection was 880.51 pg/mL, greater than that in the healthy control group (p < 0.001). One month later, the scleritis was completely resolved, and the patient remained stable during six months of follow-up. Intravitreal bevacizumab appears to be an effective adjuvant therapy for patients with posterior scleritis.

Concentration of cytokines in the aqueous humor of patients with central serous chorioretinopathy.

PURPOSE: To evaluate the levels of aqueous humor cytokines in the eyes of patients with central serous chorioretinopathy (CSC) before an intravitreal injection of bevacizumab. METHODS: In a prospective interventional trial, 20 eyes of 20 patients with symptomatic CSC of at least 3 months duration and 20 eyes of 20 patients with cataract as control were included. Eyes with CSC received a single intravitreal injection of bevacizumab (1.25 mg/0.05 mL). Aqueous humor samples were collected from patients and controls. Multiplex bead assays were used for measurement of cytokines, including vascular endothelial growth factor and platelet-derived growth factor. RESULTS: Similar vascular endothelial growth factor levels and significantly decreased platelet-derived growth factor levels were measured in the aqueous humor of eyes from patients with CSC compared with controls (P = 0.172 and P = 0.004, respectively). There was a significant inverse correlation between the vascular endothelial growth factor and platelet-derived growth factor in patients (r = -0.555; P = 0.026). Interleukin 6, interleukin 8, and monocyte chemoattractant protein-1 were detectable, but not significantly different between the patients and controls. CONCLUSION: The results of the current study suggest that platelet-derived growth factor contributes to the pathogenesis of CSC; however, the roles of vascular endothelial growth factor in CSC are unclear and warrant further investigation.

Results of spectral-domain optical coherence tomography by preferential hyperacuity perimeter in patients after idiopathic epiretinal membrane surgery.

PURPOSE: To evaluate spectral-domain optical coherence tomography (OCT) findings based on preferential hyperacuity perimeter (PHP) in patients after idiopathic epiretinal membrane surgery. METHODS: Forty eyes of 40 patients undergoing surgery for idiopathic epiretinal membrane were included in the study. The best-corrected visual acuity, PHP, Amsler grid test, and OCT were assessed 3 months after surgery. RESULTS: Patients were classified based on the defect of hyperacuity by PHP, as follows: hyperacuity defect (n = 18 eyes) and hyperacuity intact (n = 22 eyes). There was no difference in age, gender, duration of symptoms, and visual acuity. The average macular thickness and inner nuclear layer thickness in the hyperacuity defect group was thicker than the hyperacuity intact group (P = 0.041 and P = 0.045, respectively). A disrupted photoreceptor layer was more common in the hyperacuity defect group compared with the hyperacuity intact group (P = 0.001). CONCLUSION: With regard to metamorphopsia, a hyperacuity defect after epiretinal membrane surgery was associated with inner nuclear layer edema and photoreceptor disarrangement.

A comparative study between intravitreal triamcinolone and bevacizumab for macular edema due to central retinal vein occlusion with poor vision.

AIM: To compare the effect of intravitreal bevacizumab and triamcinolone in patients with macular edema after central retinal vein occlusion (CRVO), presenting with poor visual acuity. MATERIALS AND METHODS: It was a retrospective, comparative case series of 38 consecutive eyes, with macular edema secondary to CRVO, with 20/200 or worse vision, which were treated primarily either with intravitreal bevacizumab (1.25 mg; 24 eyes) or intravitreal triamcinolone (4 mg; 14 eyes). During follow-up, 3.6 ± 0.8 re-injections of bevacizumab and 2.4 ± 0.5 re-injections of triamcinolone were administered (P = 0.080). The main outcome measures were the best-corrected visual acuity and the central macular thickness by optical coherence tomography during 12 months of follow-up. RESULTS: At 12 months, visual acuity (logMAR) was changed from 1.03 ± 0.39 (baseline) to 0.92 ± 0.39 (P = 0.374) and the central macular thickness was reduced from a baseline of 713.6 ± 179.3 µm to 310.8 ± 205.2 µm (P = 0.000). Neither the bevacizumab nor triamcinolone groups varied significantly in visual acuity and central macular thickness at 1, 3, 6, and 12 months after treatment. Neovascular glaucoma developed in two of the 14 eyes (14%) in the triamcinolone group. CONCLUSION: In patients with CRVO and poor vision, intravitreal bevacizumab and intravitreal triamcinolone were associated with a reduction in macular edema; however, neither treatment achieved significant visual acuity improvement by the 12-month follow-up.

Intravitreal bevacizumab and cytokine levels in major and macular branch retinal vein occlusion.

PURPOSE: My aim was to evaluate the effect of intravitreal bevacizumab and to determine the concentrations of inflammatory cytokines in patients with macular edema due to branch retinal vein occlusion (BRVO), according to the site of the occlusion. METHODS: In a prospective interventional case series, 46 eyes of patients with macular edema due to major (n = 30) or macular BRVO (n = 16) were included. The patients were treated primarily with intravitreal bevacizumab (1.25 mg/0.05 ml) and completed 12 months of follow-up. Reinjections were performed if optical coherence tomography showed persistent or recurrent macular edema. Aqueous humor samples were collected at the time of initial intravitreal injection. Multiplex bead assays were used for the measurement of cytokines. RESULTS: The visual acuity and central macular thickness were improved significantly in patients with major and macular BRVO at 12 months. During 12 months, the eyes with major BRVO received a mean of 3.8 intravitreal injections, whereas those with macular BRVO received a mean of 1.9 intravitreal injections (p = 0.009). Significantly increased concentrations of IL-6, IL-8 and MCP-1 were observed in the aqueous humor of BRVO patients compared with control samples. Higher concentrations of IL-6, VEGF, PDGF-AA and MCP-1 were measured in the aqueous humor of patients with major BRVO compared with macular BRVO at baseline (p < 0.05). CONCLUSION: Intravitreal bevacizumab was similarly effective for improving macular edema in patients with major and macular BRVO. However, macular BRVO required fewer injections and had lower cytokine levels in the aqueous humor than major BRVO.

The efficacy of intravitreal bevacizumab for idiopathic central serous chorioretinopathy.

BACKGROUND: To evaluate the efficacy of intravitreal bevacizumab injection in the treatment of central serous chorioretinopathy (CSC). METHODS: In a prospective interventional case series, 40 eyes of 40 patients with idiopathic CSC at least 3 months in duration were included. Patients were treated with once or twice intravitreal bevacizumab injections initially and completed at least 12 months follow-up. Main outcomes measures were the resolution of neurosensory detachment, best-corrected visual acuity, and findings on fluorescein and indocyanine green angiography. RESULTS: Thirty-three of 40 eyes (82.5%) showed complete absence of subretinal fluid at the macula within 3 months following initial intravitreal bevacizumab injection. Eyes exhibiting resolution of subretinal fluid revealed improvement in fluorescein and indocyanine green angiographic findings. The rate of intense hyperfluorescence on indocyanine green angiography was higher in eyes exhibiting resolution of subretinal fluid than eyes exhibiting incomplete absorption of subretinal fluid after intravitreal bevacizumab (72.7 and 28.5%, respectively, p = 0.039). CONCLUSIONS: Intravitreal bevacizumab injections generally resulted in anatomic improvement for CSC and may constitute a therapeutic option in CSC. The effect after intravitreal bevacizumab injection for CSC might be related to the hyperfluorescence on indocyanine green angiography.

Assessment of macular function by multifocal electroretinography following epiretinal membrane surgery with internal limiting membrane peeling.

The aim of the current study was to evaluate macular function before and after surgery for idiopathic epiretinal membranes with internal limiting membrane (ILM) peeling by means of multifocal electroretinography (ERG). Eighteen eyes of 18 patients who underwent vitrectomy with ILM removal were included. Best corrected visual acuity, optical coherence tomography (OCT), and multifocal electroretinography were assessed prior to surgery, and 3 and 12 months after surgery. All surgical samples were obtained and confirmed inclusion of an ILM by electron microscopy. Visual acuity and the central foveal thickness by OCT improved significantly 3 months postoperatively, with gradual recovery by 12 months. Preoperatively, only the P1 amplitude in rings 1 and 2 were decreased compared with the normal fellow eyes (P < 0.01). Three and 12 months after surgery, the P1 amplitude in rings 1 and 2 were decreased compared with the preoperative P1 amplitudes, but without significance. The photoreceptor status by OCT was related to the N1 amplitude before and after surgery. Although visual acuity and macular edema were improved after surgery, macular function, as indicated by multifocal ERG, had limited recovery at 12 months.

Outer foveolar defect after surgery for idiopathic macular hole.

PURPOSE: To describe the clinical implications of outer foveolar defects on optical coherence tomography (OCT) in eyes treated surgically for macular hole (MH). DESIGN: Retrospective observational case series. METHODS: Ninety-six eyes of 93 subjects who had undergone surgery for the treatment of idiopathic MH were included. Clinical data, including OCT prior to and at follow-up visits ranging from 3 to 32 months after surgery, were analyzed. Incidence, risk factors, and clinical outcomes of outer foveolar defect were evaluated. RESULTS: Outer foveolar defects were noted on postoperative OCT in 44 of the 96 eyes (45.8%). The mean preoperative diameter of MH in the eyes (333.5 ± 126.3 µm) evidencing outer foveolar defects was significantly smaller than those that did not (504.2 ± 155.6 µm) (P < .0001). The mean disappearance time of the defect was estimated to be 182 days after surgery. The mean postoperative visual acuity (logMAR) improved to 0.40 ± 0.26 from 0.58 ± 0.23 after the disappearance of outer foveolar defects. CONCLUSIONS: Outer foveolar defects were detected predominantly after surgery for small MHs. The defect decreases gradually in size and eventually disappears completely approximately 6 months after surgery.

Aqueous humor and plasma levels of vascular endothelial growth factor and interleukin-8 in patients with central serous chorioretinopathy.

PURPOSE: The purpose of this study was to determine aqueous vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels in patients with central serous chori-oretinopathy (CSC) before a single intravitreal bevacizumab injection. METHODS: Twelve eyes with symptomatic CSC were included. Samples from patients with cataracts served as controls. The levels of VEGF and IL-8 concentrations were measured in aqueous humor and plasma by multiplex bead assays. RESULTS: All patients with CSC showed an improvement in visual acuity and resolved neurosensory detachment after intravitreal bevacizumab injection. The aqueous humor levels of VEGF and IL-8 were not significantly increased in patients with CSC compared with the healthy control group (18.2 ± 24.8 vs. 35.3 ± 28.5 pg/mL, P > 0.05; 2.3 ± 0.4 vs. 2.8 ± 0.3 pg/mL, P > 0.05, respectively). The plasma levels of VEGF and IL-8 in patients with CSC were not different from those in the healthy control group. CONCLUSION: Vascular endothelial growth factor and IL-8 were not increased in the aqueous humor and plasma of patients with CSC. The effect of intravitreal bevacizumab injection as a treatment for CSC must be fully understood, and the true effect of anti-VEGF treatment in patients with CSC remains to be elucidated.

The effect of intravitreal bevacizumab in patients with acute central serous chorioretinopathy.

PURPOSE: To evaluate the effect of intravitreal bevacizumab injection (IVBI) in acute central serous chorioretinopathy (CSC) patients. METHODS: Patients with acute CSC received IVBI (1.25 mg/0.05 mL) or observation by randomization. Twelve eyes in each group completed 6 months of regular follow-up and were ultimately included in this study. Each patient was assessed using best corrected visual acuity measurements, fluorescein angiography, and optical coherence tomography at baseline and had regular follow-ups after treatment. RESULTS: All patients showed improvements in visual acuity and fluorescein angiographic leakage and had resolution of their neurosensory detachment following treatment. There were no significant differences in visual acuity, central retinal thickness, or remission duration between the IVBI group and the control group at baseline or after treatment (p>0.05). CONCLUSIONS: Intravitreal bevacizumab showed no positive effect in acute CSC patients compared to the observation group, and there were no adverse effects of treatment. Further investigation will be helpful to understand this therapy in patients with CSC.