hHEPATO-Cy5, a Bimodal Tracer for Image-Guided Hepatobiliary Surgery.

Liver cancer is a leading cause of cancer deaths worldwide. Surgical resection of superficial hepatic lesions is increasingly guided by the disrupted bile excretion of the fluorescent dye indocyanine green (ICG). To extend this approach to deeper lesions, a dedicated bimodal tracer that facilitates both fluorescence guidance and radioguidance was developed. Methods: A tracer comprising a methylated cyanine-5 (Cy5) fluorescent dye and a mercaptoacetyltriserine chelate (hHEPATO-Cy5) was synthesized and characterized. Cellular uptake and excretion were evaluated in hepatocyte cultures (2-dimensional culture and in vitro lesion model), using a fluorescent bile salt, MitoTracker dye, and methylated Cy5 as a control. After radiolabeling, the pharmacokinetics of 99mTc-hHEPATO-Cy5 were assessed in mice over 24 h (percentage injected dose and percentage injected dose per gram of tissue, SPECT/CT imaging and fluorescence imaging). The ability to provide real-time fluorescence guidance during robot-assisted hepatobiliary surgery was evaluated in a porcine model using ICG as a reference. Results: The unique molecular signature of hHEPATO-Cy5 promotes hepatobiliary excretion. In vitro studies on hepatocytes showed that where methylated Cy5 remained internalized, hHEPATO-Cy5 showed fast clearance (10 min) similar to that of fluorescent bile salt. In vivo use of 99mTc-hHEPATO-Cy5 in mice revealed liver accumulation and rapid biliary clearance. The effectiveness of bile clearance was best exemplified by the 2-orders-of-magnitude reduction in count rate for the gallbladder (P = 0.008) over time. During hepatobiliary surgery in a porcine model, hHEPATO-Cy5 enabled fluorescence-based lesion identification comparable to that of ICG. Conclusion: The bimodal 99mTc-hHEPATO-Cy5 provides an effective means to identify liver lesions. Uniquely, it helps overcome the shortcomings of fluorescence-only approaches by allowing for an extension to in-depth radioguidance.

Ageing of Plasmodium falciparum malaria sporozoites alters their motility, infectivity and reduces immune activation in vitro.

BACKGROUND: Sporozoites (SPZ), the infective form of Plasmodium falciparum malaria, can be inoculated into the human host skin by Anopheline mosquitoes. These SPZ migrate at approximately 1 µm/s to find a blood vessel and travel to the liver where they infect hepatocytes and multiply. In the skin they are still low in number (50-100 SPZ) and vulnerable to immune attack by antibodies and skin macrophages. This is why whole SPZ and SPZ proteins are used as the basis for most malaria vaccines currently deployed and undergoing late clinical testing. Mosquitoes typically inoculate SPZ into a human host between 14 and 25 days after their previous infective blood meal. However, it is unknown whether residing time within the mosquito affects SPZ condition, infectivity or immunogenicity. This study aimed to unravel how the age of P. falciparum SPZ in salivary glands (14, 17, or 20 days post blood meal) affects their infectivity and the ensuing immune responses. METHODS: SPZ numbers, viability by live/dead staining, motility using dedicated sporozoite motility orienting and organizing tool software (SMOOT), and infectivity of HC-04.j7 liver cells at 14, 17 and 20 days after mosquito feeding have been investigated. In vitro co-culture assays with SPZ stimulated monocyte-derived macrophages (MoMɸ) and CD8+ T-cells, analysed by flow cytometry, were used to investigate immune responses. RESULTS: SPZ age did not result in different SPZ numbers or viability. However, a markedly different motility pattern, whereby motility decreased from 89% at day 14 to 80% at day 17 and 71% at day 20 was observed (p ≤ 0.0001). Similarly, infectivity of day 20 SPZ dropped to ~ 50% compared with day 14 SPZ (p = 0.004). MoMɸ were better able to take up day 14 SPZ than day 20 SPZ (from 7.6% to 4.1%, p = 0.03) and displayed an increased expression of pro-inflammatory CD80, IL-6 (p = 0.005), regulatory markers PDL1 (p = 0.02), IL-10 (p = 0.009) and cytokines upon phagocytosis of younger SPZ. Interestingly, co-culture of these cells with CD8+ T-cells revealed a decreased expression of activation marker CD137 and cytokine IFNγ compared to their day 20 counterparts. These findings suggest that older (day 17-20) P. falciparum SPZ are less infectious and have decreased immune regulatory potential. CONCLUSION: Overall, this data is a first step in enhancing the understanding of how mosquito residing time affects P. falciparum SPZ and could impact the understanding of the P. falciparum infectious reservoir and the potency of whole SPZ vaccines.