Daniele de Paula Faria

The Brazilian Zika virus strain causes birth defects in experimental models

Summary Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (Family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys1. Until the 20th century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the island of Yap in Micronesia2. Patients experienced fever, skin rash, arthralgia and conjunctivitis2. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America3. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome4,5. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKVBR) strain causes birth defects remains missing6. Here we demonstrate that the ZIKVBR infects fetuses, causing intra-uterine growth restriction (IUGR), including signs of microcephaly in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. Finally, we observed that the infection of human brain organoids resulted in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKVBR crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKVBR outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKVBR in human neurodevelopment.

PET imaging of demyelination and remyelination in the cuprizone mouse model for multiple sclerosis: a comparison between [11C]CIC and [11C]MeDAS.

Multiple Sclerosis (MS) is a neurodegenerative disease characterized by demyelinated lesions. PET imaging using specific myelin radioligands might solve the lack of a specific imaging tool for diagnosing and monitoring demyelination and remyelination in MS patients. In recent years, a few tracers have been developed for in vivo PET imaging of myelin, but they have not been fully evaluated yet. In this study, we compared [(11)C]CIC and [(11)C]MeDAS as PET tracers for monitoring demyelination and remyelination in cuprizone-fed mice. The ex vivo biodistribution of [(11)C]CIC showed decreased tracer uptake in mice fed with 0.2% cuprizone diet for 5 weeks, as compared to control mice. However, tracer uptake did not increase again after normal diet was restored for 5 weeks (remyelination). Surprisingly, in vivo PET imaging with [(11)C]CIC in cuprizone-fed mice revealed a significant reduction in whole brain tracer uptake after 5 weeks of remyelination. No correlation between ex vivo biodistribution and in vivo imaging data was found for [(11)C]CIC (r(2)=0.15, p=0.11). However, a strong correlation was found for [(11)C]MeDAS (r(2)=0.88, p<0.0001). [(11)C]MeDAS ex vivo biodistribution revealed significant decreased brain uptake in the demyelination group, as compared to controls and increased the tracer uptake after 5 weeks of remyelination. [(11)C]MeDAS images showed a low background signal and clear uptake in the brain white matter and spinal cord. Taken together, the results of this comparative study between [(11)C]CIC and [(11)C]MeDAS clearly show that [(11)C]MeDAS is the preferred PET tracer to monitor myelin changes in the brain and spinal cord in vivo.

PET imaging in multiple sclerosis

Positron emission tomography (PET) is a non-invasive technique for quantitative imaging of biochemical and physiological processes in animals and humans. PET uses probes labeled with a radioactive isotope, called PET tracers, which can bind to or be converted by a specific biological target and thus can be applied to detect and monitor different aspects of diseases. The number of applications of PET imaging in multiple sclerosis is still limited. Clinical studies using PET are basically focused on monitoring changes in glucose metabolism and the presence of activated microglia/macrophages in sclerotic lesions. In preclinical studies, PET imaging of targets for other processes, like demyelination and remyelination, has been investigated and may soon be translated to clinical applications. Moreover, more PET tracers that could be relevant for MS are available now, but have not been studied in this context yet. In this review, we summarize the PET imaging studies performed in multiple sclerosis up to now. In addition, we will identify potential applications of PET imaging of processes or targets that are of interest to MS research, but have yet remained largely unexplored.

The contributions of dipeptidyl peptidase IV to inflammation in heart failure

Circulating dipeptidyl peptidase IV (DPPIV) activity correlates with cardiac dysfunction in humans and experimental heart failure (HF) models. Similarly, inflammatory markers are associated with poorer outcomes in HF patients. However, the contributions of DPPIV to inflammation in HF remain elusive. Therefore, this study aimed to investigate whether the cardioprotective effects of DPPIV inhibition after myocardial injury are accompanied by reduced cardiac inflammation, whether circulating DPPIV activity correlates with the levels of systemic inflammatory markers in HF patients, and whether leukocytes and/or splenocytes may be one of the sources of circulating DPPIV in HF. Experimental HF was induced in male Wistar rats by left ventricular myocardial injury after radiofrequency catheter ablation. The rats were divided into three groups: sham, HF, and HF + DPPIV inhibitor (sitagliptin). Six weeks after surgery, cardiac function, perfusion and inflammatory status were evaluated. Sitagliptin treatment improved cardiac function and perfusion, reduced macrophage infiltration, and diminished the levels of inflammatory biomarkers including TNF-α, IL-1β, and CCL2. In HF patients, serum DPPIV activity correlated with CCL2, suggesting that leukocytes may be the source of circulating DPPIV in HF. Unexpectedly, DPPIV release was higher in splenocytes from HF rats and similar in HF circulating mononuclear cells compared with those from sham, suggesting an organ-specific modulation of DPPIV in HF. Collectively, our data provide new evidence that the cardioprotective effects of DPPIV inhibition in HF may be due to suppression of inflammatory cytokines. Moreover, they suggest that a vicious circle between DPPIV and inflammation may contribute to HF development and progression.

PET Imaging of Disease Progression and Treatment Effects in the Experimental Autoimmune Encephalomyelitis Rat Model

The experimental autoimmune encephalomyelitis model is a model of multiple sclerosis that closely mimics the disease characteristics in humans. The main hallmarks of multiple sclerosis are neuroinflammation (microglia activation, monocyte invasion, and T-cell infiltration) and demyelination. PET imaging may be a useful noninvasive technique for monitoring disease progression and drug treatment efficacy in vivo. Methods: Experimental autoimmune encephalomyelitis was induced by myelin-oligodendrocyte glycoprotein immunization in female Dark Agouti rats. Experimental autoimmune encephalomyelitis rats were imaged at baseline and at days 6, 11, 15, and 19 after immunization to monitor monocyte and microglia activation (11C-PK11195) and demyelination (11C-MeDAS) during normal disease progression and during treatment with dexamethasone. Results: 11C-PK11195 PET detected activation of microglia and monocytes in the brain stem and spinal cord during disease progression. The uptake of 11C-PK11195 was elevated in dexamethasone-treated animals that had shown mild clinical symptoms that had resolved at the time of imaging. Demyelination was not detected by 11C-MeDAS PET, probably because of the small size of the lesions (average, 0.13 mm). Conclusion: PET imaging of neuroinflammation can be used to monitor disease progression and the consequences of treatment in the experimental autoimmune encephalomyelitis rat model. PET imaging was more sensitive than clinical symptoms for detecting inflammatory changes in the central nervous system.

Evaluation of exercise-induced modulation of glial activation and dopaminergic damage in a rat model of Parkinson’s disease using [11C]PBR28 and [18F]FDOPA PET:

Evidence suggests that exercise can modulate neuroinflammation and neuronal damage. We evaluated if such effects of exercise can be detected with positron emission tomography (PET) in a rat model of Parkinson’s disease (PD). Rats were unilaterally injected in the striatum with 6-hydroxydopamine (PD rats) or saline (controls) and either remained sedentary (SED) or were forced to exercise three times per week for 40 min (EX). Motor and cognitive functions were evaluated by the open field, novel object recognition, and cylinder tests. At baseline, day 10 and 30, glial activation and dopamine synthesis were assessed by [11C]PBR28 and [18F]FDOPA PET, respectively. PET data were confirmed by immunohistochemical analysis of microglial (Iba-1) / astrocyte (GFAP) activation and tyrosine hydroxylase (TH). [11C]PBR28 PET showed increased glial activation in striatum and hippocampus of PD rats at day 10, which had resolved at day 30. Exercise completely suppressed glial activation. Imaging results correlated well with post-mortem Iba-1 staining, but not with GFAP staining. [18F]FDOPA PET, TH staining and behavioral tests indicate that 6-OHDA caused damage to dopaminergic neurons, which was partially prevented by exercise. These results show that exercise can modulate toxin-induced glial activation and neuronal damage, which can be monitored noninvasively by PET.

18F-Fluoride PET/CT and 99mTc-MDP SPECT/CT can detect bone cancer at early stage in rodents

&NA; Noninvasive imaging using positron emission tomography/computed tomography (PET/CT) and single photon emission computed tomography/computed tomography (SPECT/CT) are considered revolutionized approaches to detect bone cancer. Both PET/CT and SPECT/CT technologies have advanced to permit miniaturization, which has provided the advantage of including animals as their own controls in longitudinal studies. The present study was designed to evaluate the potential of PET/CT and SPECT/CT as research tools to detect bone cancer in rats. We used a rat model of bone cancer induced by injecting Walker 256 tumor cells into the femoral cavity. Computed tomography demonstrated that rats presented a solid tumor at 15 days post injection (dpi). However, CT was not an effective method for identifying tumors at an earlier time point (8 dpi), when mechanical hyperalgesia (the most common symptom during bone cancer progression) had already initiated. At this early stage, PET/CT and SPECT/CT analysis detected higher uptake in the injected femur of the tracers 18F‐Fluoride and 99mTc‐Methyl diphosphonate (99mTc‐MDP), respectively. These findings demonstrated for the first time that both 18F‐Fluoride PET/CT and 99mTc‐MDP SPECT/CT can detect cancer at early stages in rats and advocates for the PET/SPECT/CT as research tools to evaluate bone cancer in further longitudinal studies involving small animals.

[18F]FDG PET imaging evaluation on non-alcoholic fatty liver disease and hepatocellular carcinoma model treated with sorafenib

Aim: Evaluate the effect of sorafenib in a rat model of non-alcoholic fatty liver disease (NAFLD) related to hepatocellular carcinoma (HCC) by quantifying the correlation between changes in glucose metabolism on PET imaging and degree of tumor differentiation. Methods: NAFLD related HCC was induced by the combination of high fat and choline deficient diet with diethylnitrosamine (100 mg/L) for 16 weeks. Then carcinogenic stimuli were suspended, liver nodules were identified by abdominal ultrasound and two groups were randomized: control (n = 10) and sorafenib (n = 20). Rats received daily gavage administration of 1 mL saline or sorafenib (5 mg/kg/day) for more 3 weeks. After treatment, [F]FDG PET scan was performed on animals. Results: [F]FDG uptake was lower in the sorafenib group than that in the control group (3.3 ± 0.48 vs . 5.5 ± 1.5, P = 0.01). Direct correlation was found between poorly-differentiated HCC and TumorSUVmax/MuscleSUVmax ratio (R = 0.54, P = 0.006). Treatment was associated with significantly more residual tumors that were well differentiated (Grades I/II) than in the untreated control group (39% vs . 5%, respectively, P = 0.01). Conclusion: Sorafenib shows promise as a treatment for reducing the aggressiveness of HCC as demonstrated by [F] FDG PET and immunohistochemistry.

7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma.

Background: The exposure of phosphatidylserine (PS) is one of the first steps of programmed cell death. Phagocytosis on cancer microenvironment is well described in tumors and is associated with malignancy and poor prognosis. Tumor associated macrophages (TAMs) act suppressing the anticancer immune response. The tumor parenchymal cells are also capable of phagocytosis cells in apoptosis. In a previous study we observed that 7-ketocholesterol is capable of inducing autophagy on melanoma cell. Aims: Evaluate the activities of a 7-ketocholesterol loaded-phosphatidylserine liposome on autophagy and phagocytosis of tumor microenvironment. Methods: Liposomes were constituted by 20 mg commercial Phosphatidylserine (PS) and PS associated with 5 mg of 7-ketocholesterol extracted with chloroform/methanol (10: 1), dried, resuspended in 10 mL phosphate buffer, homogenized and sonicated for 6 minutes. The size and Zeta Pontencial (ZP) of liposomes were evaluated. Antiinflammatory activity of liposomes was evaluated by paw edema induced by carrageenan. A dependent-dose effect of liposomes on J774 macrophages, B16F10 melanoma cells, and 4T1 breast cancer cells was assessed by MTT. Cell death evaluations, for the same cells, were performed by flow cytometry with propidium iodide (PI) staining. The presence of acid vacuoles related to autophagy was evaluated by flow cytomery by acridine orange staining. The effects of the liposomes in vivo were evaluated by B16F10 melanoma-bearing C57/bl6 mice and 4T1 breast cancer-bearing Balb c mice. Endocytosis efficiency of the liposomes was observed by labeling it with PKH26 fluorescent staining and evaluated in 4T1 cells after 12 h. Liposomes were radiolabeled by adding 1 to 30 mCi of 99mTc radiopharmaceuticals (99mTcO4-, 99mTc-dextran-70, 99mTc-MIBI, 99mTc-DISIDA) and 18FDG; the solution was homogenized and sonicated for 6 minutes. The samples were centrifuged and part of the supernatant was added to an Amicon® filter (10kD) and concentrated, the concentrated was diluted with 400 uL of PBS and concentrated again. Liposome incorporation was determined by quotient of the radioactivity in the Amicon® by sum of Amicon® and filtrated solutions. Furthermore, lipophilicity (L), hydrophilicity (H), and charge (-/0/+) of the radioactive material were considered in the final analysis. Results: PS liposomes presented 141,9nm + 9,101 size with a -25,2 ZP; PS-7-ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35 size with a -29,1 ZP. The paw edema was inhibited by both liposomes after 240 min of the carrageenan induction. The concentration of 26 uM/mL of PS and PS/7KC liposomes stimulated cell proliferation. PS/7KC at the concentrations above 84 uM/mL inhibited the cell proliferation. PS/7KC showed intense antiproliferative activity in melanoma cells and breast adenocarcinoma cells, assessed by the MTT method and by flow cytometry with PI. It was observed 10% more autophagic vacuoles on melanoma cells treated with PS/7KC than the control groups. Both in vivo tumor models had the same antiproliferative effect of the PS/7KC liposomes with daily doses. Daily doses of PS liposomes induced a high size of tumors. 99mTc-MIBI was efficiently and strongly incorporated to liposomes than the other proposed formulations. Conclusion: PS liposomes have effects in vivo and in vitro and must be related to phagocyte and autophagy activities. PS/7KC impairs J774 macrophage, B16F10 melanoma, and 4T1 breast adenocarcinoma cell growth. PS/7KC induces the presence of acid vacuoles corresponding to autophagy. The liposomes had a high endocytosis evaluated by PKH 26 labelled particles. PS keeps the tumor proliferation and PS/7KC inhibits tumor growth after ten days of daily doses. Supported by CNPq and Fundacao Araucaria. Citation Format: Giovani Marino Favero, Tharcisio Citrangulo Tortelli, Jr., Daniel Fernandes, Ana Paula Prestes, Louise N.B. Kmetiuk, Andreia Hanada Otake, Luciana N.S. Andrade, Daniele de Paula Faria, Camila de Godoi Carneiro, Alexandre Teles Garcez, Fabio L.N. Marques, Roger Chammas. 7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A50.

Alternative chromatographic system for the quality control of lipophilic technetium-99m radiopharmaceuticals such as [99mTc(MIBI)6]+

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [99mTc(MIBI)6]+ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [99mTc(MIBI)6]+ since impurities such as 99mTc-reduced-hydrolyzed (RH), 99mTcO4 - and [99mTc(cysteine)2]- complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [99mTc(MIBI)6]+ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [99mTc(MIBI)6]+ since impurities such as99mTc-reduced-hydrolyzed (RH),99mTcO4 - and [99mTc(cysteine)2]-complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.