Near-infrared labeled,ovalbumin loaded polymeric nanoparticles based on a hydrophilic polyester as model vaccine: In vivo tracking and evaluation of antigen-specific CD8+ T cell immune response

Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co-hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300–400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice. We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8+ T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.     

载吲哚菁绿超声微泡造影剂活体近红外荧光显像兔淋巴结

目的制备载吲哚菁绿的聚乳酸羟基乙酸（ICG-PLGA）超声微泡造影剂并检测其特性,观察以之进行活体近红外荧光显像兔淋巴结的效果。方法用双乳化法制备ICG-PLGA超声微泡造影剂,行光镜、扫描电镜、粒径检测检查,观察其形态及大小;用分光光度法绘制吲哚菁绿的标准曲线,计算造影剂中吲哚菁绿的载药量和包封率。对3只正常大白兔经足垫注射该造影剂,观察活体近红外荧光显像兔腘窝淋巴结的效果。结果 ICG-PLGA超声微泡造影剂为淡绿色乳液,光镜和电镜下形态规则,表面光滑,大小均匀;活体近红外荧光成像能清晰显示兔腘窝淋巴结。结论 ICG-PLGA超声微泡造影剂具备荧光成像的特征,具有高敏感性,可作为荧光成像的示踪剂。     

A feasibility study of cerebral oximetry monitoring during the post-resuscitation period in comatose patients following cardiac arrest

Background

One of the major causes of death and neurological injury after cardiac arrest is delayed ischemia combined with oxygen free radical mediated reperfusion injury. Consequently determining the optimal balance between oxygen delivery and uptake in the brain using a reliable non-invasive monitoring system during the post-resuscitation period is of importance. In this observational study, we evaluated the feasibility of using cerebral oximetry during the post-resuscitation period in order to identify changes in regional cerebral oxygen saturation (rSO₂) and its association with survival to discharge.

Methods

21 consecutive patients admitted to the intensive care units following cardiac arrest had cerebral oximetry monitoring carried out for 48 h. Mean rSO₂ values were collected during the first 24 h and then again during the subsequent 24–48 h of the post-resuscitation period.

Results

43% (n = 9) patients survived to hospital discharge and 57% (n = 12) died. Amongst all patients the median (IQR) rSO₂% was 65.5% (62.6–68.2) in the first 24-h following ROSC and increased to 72.1% (64.6–73.7) (p = 0.11) in the subsequent 24–48 h. The median (IQR) rSO₂% during the first 24 h in patients who survived to discharge compared to those who did not survive were significantly higher 68.2% (66.0–71.0) vs. 62.9% (56.5–66.0), p = 0.01). During the subsequent 24–48 h period, while a difference in the rSO₂ between survivors and non-survivors was noted, this did not achieve statistical significance (median (IQR): 73.7 (70.2–74.0) vs. 66.5 (58.2–72.1), p = 0.11).

Conclusions

Our study indicates that the use of cerebral oximetry is feasible during the post resuscitation period after cardiac arrest. Further studies are needed to determine whether cerebral oximetry may be used as a novel non-invasive monitoring system to evaluate changes in the balance between cerebral oxygen delivery and uptake during the post-resuscitation period.     

Near Infrared Spectroscopy Monitoring During Carotid Endarterectomy: Which Threshold Value is Critical?

OBJECTIVES: Retrospectively to verify which decreasing percentage in regional oxygen saturation (rSO(2)) identified patients with good collateralisation during carotid artery cross clamp. MATERIALS AND METHODS: During 594 endarterectomies under general anaesthesia the decreasing percentage from preclamp value to value detected in the first 2 min after clamping the CCA and/or ICA was calculated in real time. No temporary shunt was placed in any case. ROC analysis was performed to determine the optimal cut-off for rSO(2) decrease to identify the occurrence of neurological complications. RESULTS: A cut-off of 11.7% was identified as optimal. Sensitivity and specificity were 75% (95% CI 71-78) and 77% (95% CI 74-80), respectively. The cut-off of 20% had a lower sensitivity (30%) and a higher specificity (98%) to identify patients with complications, with positive and negative predictive value of 37 and 98%, respectively. CONCLUSIONS: The study suggest that a relative decrease in rSO(2) of <20% from preclamp to early cross clamp value has a high negative predictive value, i.e. if rSO(2) does non decrease more than 20%, ischemia by hypoperfusion is unlikely and a shunt should not be necessary. Moreover, a relative decrease >20% may not always indicate intraoperative neurological complications.     

Suitability of near-infrared methods for the determination of moisture in a freeze-dried injection product containing different amounts of the active ingredient

A near-infrared reflectance (NIR) method for determination of moisture in an experimental freeze-dried injection product was developed and validated. NIR spectra were collected through the bases of unopened product vials using a horizontal instrument accessory, before generating primary reference data on the same individual vials by Karl-Fischer titration. Data were collected for product containing different concentrations of the active ingredient in the same matrix. NIR calibrations were developed with second derivative spectral data using regression facilities within the NIR software, and validated using independent test sets. An assessment is given of the applicability of moisture calibrations developed on product at one active ingredient level to the prediction of moisture contents in the product containing a different concentration of active ingredient.     

Sentinel Lymph Node Mapping of the Gastrointestinal Tract by Using Invisible Light

Background Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. Methods We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. Results In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. Conclusions We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.     

Image-Guided Oncologic Surgery Using Invisible Light: Completed Pre-Clinical Development for Sentinel Lymph Node Mapping

Background Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma.Methods Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time.Results Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo.Conclusions This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.     

Reliability of Near-Infrared Spectroscopy in People With Dark Skin Pigmentation

Objective. Near-infrared spectroscopy (NIRS) is a promising non-invasive technique for the continuous monitoring of tissue oxygen delivery. NIRS detects light absorbance of haemoglobin chromophores to determine tissue oxygen saturation (StO₂). As skin colour is also determined by the presence of chromophores, it is plausible that NIRS signal quality may be affected by dark skin pigmentation. Methods. Tissue saturation in the anterior compartment of the lower leg during isometric contraction was measured using NIRS in 17 volunteers with dark skin pigmentation. Measurements were continued until StO2 was zero percent or until the signal disappeared. Results. The NIRS device failed to register tissue saturation values at some point in nine of seventeen volunteers. This occurred more often in individuals with darker skin. Conclusions. In patients with a dark pigmented skin, NIRS StO₂ measurements should be interpreted with caution, as melanin clearly interferes with the quality of the reflected NIRS signal.