Abstract A brief exposure of skin to a low-power, non-tissue damaging laser light has been demonstrated to augment immune responses to intradermal vaccination. Both preclinical and clinical studies ...show that this approach is simple, effective, safe and well tolerated compared to standard chemical or biological adjuvants. Until now, these laser exposures have been performed using a diode-pumped solid-state laser (DPSSL) devices, which are expensive and require labor-intensive maintenance and special training. Development of an inexpensive, easy-to-use and small device would form an important step in translating this technology toward clinical application. Here we report that we have established a handheld, near-infrared (NIR) laser device using semiconductor diodes emitting either 1061, 1258, or 1301 nm light that costs less than $4000, and that this device replicates the adjuvant effect of a DPSSL system in a mouse model of influenza vaccination. Our results also indicate that a broader range of NIR laser wavelengths possess the ability to enhance vaccine immune responses, allowing engineering options for the device design. This small, low-cost device establishes the feasibility of using a laser adjuvant approach for mass-vaccination programs in a clinical setting, opens the door for broader testing of this technology with a variety of vaccines and forms the foundation for development of devices ready for use in the clinic.
Efficient and timely delivery of vaccine antigens to the secondary lymphoid tissue is crucial to induce protective immune responses by vaccination. However, determining the longitudinal ...biodistribution of injected vaccines in the body has been a challenge. Here, the near‐infrared (NIR) fluorescence imaging is reported that can efficiently enable the trafficking and biodistribution of vaccines in real time. Zwitterionic NIR fluorophores are conjugated on the surface of model vaccines and tracked the fate of bioconjugated vaccines after intradermal administration. Using an NIR fluorescence imaging system, it is possible to obtain time‐course imaging of vaccine trafficking through the lymphatics, observing notable uptake in lymph nodes with minimal nonspecific tissue interactions. Flow cytometry analysis confirmed that the uptake in lymph nodes by antigen presenting cells was highly dependent on the hydrodynamic diameter of vaccines. These results demonstrate that the combination of a real‐time NIR fluorescence imaging system and zwitterionic fluorophores is a powerful tool to determine the fate of vaccine antigens. Since such non‐specific vaccine uptake causes serious adverse reactions, this method is not only useful for optimization of vaccine design, but also for safety evaluation of clinical vaccine candidates.
Biodistribution of vaccines is reliably monitored using a renal clearable zwitterionic near‐infrared (NIR) fluorophore with a high signal‐to‐background ratio in vivo. The size‐dependent transportation of vaccine from injection sites to the secondary lymphoid tissues is reproducibly described using a multispectral NIR imaging platform, showing the applicability of this approach to optimize formulation and evaluate the safety of vaccines.
Brief exposure of skin to near-infrared (NIR) laser light has been shown to augment the immune response to intradermal vaccination and thus act as an immunologic adjuvant. Although evidence indicates ...that the NIR laser adjuvant has capacity to activate innate subsets including dendritic cells (DCs) in skin as conventional adjuvants do, the precise immunological mechanism by which the NIR laser adjuvant acts is largely unknown.
Here we sought to identify the cellular target of the NIR laser adjuvant by using an established mouse model of intradermal influenza vaccination and examining the alteration of responses resulting from genetic ablation of specific DC populations. We found that a continuous wave (CW) NIR laser adjuvant broadly modulates migratory DC populations, specifically increasing and activating the Lang
+
and CD11b
−
Lang
−
subsets in skin, and that the antibody responses augmented by the CW NIR laser are dependent on DC subsets expressing CCR2 and Langerin. In comparison, a pulsed wave (PW) NIR laser adjuvant showed limited effects on the migratory DC subsets. Our vaccination study demonstrated that the efficacy of CW NIR laser is significantly better than that of PW laser, indicating that the CW NIR laser offers a desirable immunostimulatory microenvironment for migratory DCs.
These results demonstrate the unique ability of the NIR laser adjuvant to selectively target specific migratory DC populations in skin depending on its parameters, and highlight the importance of optimization of laser parameters for desirable immune protection induced by a NIR laser-adjuvanted vaccine.
Nonalcoholic fatty liver disease (NAFLD) is a common disease of chronic liver diseases. Peroxisome proliferator-activated receptor α (PPARα) has been implicated to play important roles in the ...development of the disease. Beyond its effects on lipid metabolisms, PPARα activation in the vascular system has emerged as an attractive therapeutic potential for NAFLD, although its actions in the microcirculatory system are not fully understood. In this study, we investigated the effects of fenofibrate, a PPARα synthetic agonist, on hepatic microcirculation in a high-fat diet (HFD)-induced fatty liver in mice. In vivo imaging analysis revealed the adverse effects of HFD on hepatic vasculature with narrowing of hepatic sinusoids and hepatic microcirculatory perfusion. Oxygen tension was significantly decreased in portal venules, while NADH autofluorescence in hepatocytes was greatly elevated. Fenofibrate treatment remarkably improved microvascular patency, tissue oxygenation and redox states in the affected liver. These results suggest beneficial roles of PPARα activated by fenofibrate on the regulation of both lipid metabolisms and microvascular environments of oxygen metabolism in HFD-induced fatty liver.
Carbon monoxide (CO) is a gaseous mediator, which is generated via anenzymatic reaction of heme oxygenase, and it plays physiological roles in regulating cellular respiration and blood flow in the ...liver. The concentration and distribution of CO molecules in the living body is unknown owing to a lack of a suitable technique for measuring them in vivo. A needle-type CO sensor has been used for bioinstrumentation, but it is inappropriate for implantation in vivo and long-term monitoring. We developed a CO sensor sheet based on hemoglobin (Hb) allostery, as Hb undergoes a conformational change on CO binding. Hb was extracted from mice blood and mixed with agarose gel with a reducer to stabilize deoxy-Hb in the gel. CO-releasing molecules (CORM) were used to mimic CO-generating tissue, and the sensitivity of the Hb gel could be regulated by Hb concentration. We defined the CO-Hb index, an absorbance ratio at 539 and 557 nm, to estimate the accumulation of captured CO in the gel. It correlatively increased with CORM dose, indicating that gel-embedded Hb underwent a conformational change on CO binding, thereby acting as a CO sensor. We subsequently used the Hb-sensor sheet for two-dimensional imaging of CO distribution. CORM-containing gels with different sizes and doses were layered on this sheet. Size- and dose-dependent CO distribution was visualized by scanning the CO-Hb index in the sheet. Our Hb-based CO sensor sheet is composed of biocompatible materials and can be applied to detect low-level CO sources in the living body.
A catheter-type optical oxygen sensor based on phosphorescence lifetime was developed for medical and animal experimental use. Since the sensor probe should have biocompatibility and high oxygen ...permeability in vivo, we focused attention on acceptable polymer materials for contact lenses as the substrates of probes. Pd-porphyrin was doped in silicone-based polymer, and was fixed at the edge of an optical fiber inserted in a catheter tube. The shape of the probe was 600 μm in diameter and 100 μm in thickness, and the probe had high oxygen permeability of Dk value 455. In accuracy evaluation, there found an excellent correlation between the pO
2 values measured through phosphorescence lifetime using the oxygen sensors and those measured as the calibrating data using oxygen electrodes. The response time required to achieve 90% from reversible default value to be from 150 to 0 mmHg, and from 0 to 150 mmHg was 15.43 and 7.52 s, respectively. In addition, other properties such as temperature and pH dependency, response, and durability of our optical oxygen sensor were investigated. In animal experiments, the catheter-type oxygen sensor was inserted via the femoral artery of a rat, and arterial oxygen pressure was monitored under asphyxiation. The sensor was valid in the range of oxygen concentration sufficient for biometry, and expected to be integrated with an indwelling needle.
Abstract
Background: Abnormal blood vessels in solid tumors hinder the delivery and efficacy of therapeutic agents. We and others have demonstrated that these physiological barriers can be overcome ...by normalization of the vascular (Science 307: 58, 2005). We have also reported that nitric oxide (NO) produced by endothelial NO synthase (eNOS) in vascular endothelial cells mediates angiogenesis and vessel maturation while ectopic NO production by tumor cells impairs tissue NO distribution and interferes with these processes (Nature Medicine 14:255, 2008). Inducible NO synthase (iNOS) is the most frequently expressed NOS in human tumors including breast cancers. Tumor hypoxia can also induce iNOS. Resulting heterogeneous NO distribution may contribute to the abnormality of tumor vasculature. Here, we determined the role of iNOS coming from tumor and/or stromal cells on the tumor vasculature and response to radiation therapy.
Methods: Murine breast cancers (MCaIV and E0771) were grown orthotopically in the mammary fat pad in C3H and C57BL/6 mice, respectively. Perivascular cells were visualized using α-smooth muscle actin promoter driven green fluorescent protein or DsRed reporter mice. Tissue oxygenation was determined by a redox marker pimonizadole. To block iNOS in these tumors, we 1) silenced iNOS expression in tumor cells by shRNA, 2) used iNOS deficient mice to assess the role of iNOS in host stromal cells, and 3) used an iNOS selective inhibitor 1400W (10mg/kg/day). Fractionated radiation therapy (5 Gy/day × 3 days) was performed when mammary fat pad tumors reached 100 mm3.
Results: Murine breast cancers express high levels of iNOS especially in vivo. The majority of iNOS expressing cells were tumor cells. Blood vessels have eNOS but not iNOS. Both genetic and pharmacological inhibition of iNOS resulted in an increased number of blood vessels (segments) and a decrease in the abnormal vessel dilation in the breast tumors. Furthermore, iNOS inhibition increased vessel maturation and suppressesd vascular hyperpermeability. Finally, iNOS silencing and inhibition decreased hypoxia and thus, improved tumor response to radiation therapy. In contrast, these parameters were not different between iNOS deficient and wild-type mice suggesting tumor cell-derived NO dominancy.
Conclusion: In this study, we found that restoration of tissue distribution of NO in murine breast cancers to a normal pattern could normalize tumor vasculature both morphologically and functionally. The perivascular NO gradient significantly alleviated tissue hypoxia and improved efficacy of radiation therapy. Our data suggest that aberrant production of NO from tumor cells interfere with vessel maturation, and that restoration of tissue NO distribution by selective NOS modulation can improve tumor vascular function and facilitate concomitantly administered cytotoxic therapies. (Supported by NIH grant CA096915)
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-288. doi:10.1158/1538-7445.AM2011-LB-288
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