Ultra-high-resolution 3D imaging of atherosclerosis in mice with synchrotron differential phase contrast: a proof of concept study.

The goal of this study was to investigate the performance of 3D synchrotron differential phase contrast (DPC) imaging for the visualization of both macroscopic and microscopic aspects of atherosclerosis in the mouse vasculature ex vivo. The hearts and aortas of 2 atherosclerotic and 2 wild-type control mice were scanned with DPC imaging with an isotropic resolution of 15 μm. The coronary artery vessel walls were segmented in the DPC datasets to assess their thickness, and histological staining was performed at the level of atherosclerotic plaques. The DPC imaging allowed for the visualization of complex structures such as the coronary arteries and their branches, the thin fibrous cap of atherosclerotic plaques as well as the chordae tendineae. The coronary vessel wall thickness ranged from 37.4 ± 5.6 μm in proximal coronary arteries to 13.6 ± 3.3 μm in distal branches. No consistent differences in coronary vessel wall thickness were detected between the wild-type and atherosclerotic hearts in this proof-of-concept study, although the standard deviation in the atherosclerotic mice was higher in most segments, consistent with the observation of occasional focal vessel wall thickening. Overall, DPC imaging of the cardiovascular system of the mice allowed for a simultaneous detailed 3D morphological assessment of both large structures and microscopic details.

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway.

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.

Biological Characterization of Gene Response to Insulin-Induced Hypoglycemia in Mouse Retina.

Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH) decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery) on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining "normal" GSH level, as well as a strict glycemic control, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.

A new method for intraoperative localization of epilepsy focus by means of a gamma probe

Objective To evaluate the utility of a new multimodal image-guided intervention technique to detect epileptogenic areas with a gamma probe as compared with intraoperative electrocorticography. Materials and Methods Two symptomatic patients with refractory epilepsy underwent magnetic resonance imaging, videoelectroencephalography, brain SPECT scan, neuropsychological evaluation and were submitted to gamma probe-assisted surgery. Results In patient 1, maximum radioactive count was initially observed on the temporal gyrus at about 3.5 cm posteriorly to the tip of the left temporal lobe. After corticotomy, the gamma probe indicated maximum count at the head of the hippocampus, in agreement with the findings of intraoperative electrocorticography. In patient 2, maximum count was observed in the occipital region at the transition between the temporal and parietal lobes (right hemisphere). During the surgery, the area of epileptogenic activity mapped at electrocorticography was also delimited, demarcated, and compared with the gamma probe findings. After lesionectomy, new radioactive counts were performed both in the patients and on the surgical specimens (ex-vivo). Conclusion The comparison between intraoperative electrocorticography and gamma probe-assisted surgery showed similarity of both methods. The advantages of gamma probe include: noninvasiveness, low cost and capacity to demonstrate decrease in the radioactive activity at the site of excision after lesionectomy.

A química medicinal de N-acilidrazonas: novos compostos-protótipos de fármacos analgésicos, antiinflamatórios e anti-trombóticos

In this article are described new bioactive N-acylhydrazone (NAH) derivatives, structurally designed as optimization of aryl hydrazones precursors planned by molecular hybridization of two 5-lipoxigenase inhibitors, e.g. CBS-1108 and BW-755c. The analgesic, antiedematogenic and anti-platelet aggregating profile of several isosteric compounds was investigated by using classic pharmacological assays in vivo and ex-vivo, allowing to identify new potent peripheric analgesic lead, a new anti-inflammatory and an antithrombotic agent. During this study was discovered dozen of active NAH compounds clarifying the structure-activity relationship for this series of NAH derivatives, indicating the pharmacophore character of the N-acylhydrazone functionality.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rhein-huprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and (-)-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and (-)-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and (-)-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rhein-huprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and (-)-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and (-)-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and (-)-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rhein-huprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and (-)-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and (-)-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and (-)-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Imaging of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of PET Tracers for Vascular Inflammation

Atherosclerosis is a vascular inflammatory disease causing coronary artery disease, myocardial infarct and stroke, the leading causes of death in Finland and in many other countries. The development of atherosclerotic plaques starts already in childhood and is an ongoing process throughout life. Rupture of a plaque and the following occlusion of the vessel is the main reason for myocardial infarct and stroke, but despite extensive research, the prediction of rupture remains a major clinical problem. Inflammation is considered a key factor in the vulnerability of plaques to rupture. Measuring the inflammation in plaques non-invasively is one potential approach for identification of vulnerable plaques. The aim of this study was to evaluate tracers for positron emission tomography (PET) imaging of vascular inflammation. The studies were performed with a mouse model of atherosclerosis by using ex vivo biodistribution, autoradiography and in vivo PET and computed tomography (CT). Several tracers for inflammation activity were tested and compared with the morphology of the plaques. Inflammation in the atherosclerotic plaques was evaluated as expression of active macrophages. Systematic analysis revealed that the uptake of 18F-FDG and 11C-choline, tracers for metabolic activity in inflammatory cells, was more prominent in the atherosclerotic plaques than in the surrounding healthy vessel wall. The tracer for αvβ3 integrin, 18Fgalacto- RGD, was also found to have high potential for imaging inflammation in the plaques. While 11C-PK11195, a tracer targeted to receptors in active macrophages, was shown to accumulate in active plaques, the target-to-background ratio was not found to be ideal for in vivo imaging purposes. In conclusion, tracers for the imaging of inflammation in atherosclerotic plaques can be tested in experimental pre-clinical settings to select potential imaging agents for further clinical testing. 18F-FDG, 18F-galacto-RGD and 11C-choline choline have good properties, and further studies to clarify their applicability for atherosclerosis imaging in humans are warranted.

Targeted Therapy Possibilities for Metastatic Melanoma

The incidence of malignant melanoma of the skin has been steadily rising worldwide during the past decades. Most early detected primary tumors can be removed surgically and the prognosis is good. However, at the same time there still is no permanent cure for metastatic melanoma and its prognosis is poor, although lately new effective drugs have emerged. In this thesis, four different approaches of experimental therapy for metastatic melanoma were studied. Endogenous cis-Urocanic acid (UCA) is found in every individual’s skin, where exposure to UV light from the sun generates it from its inactive trans conformation. Cis- UCA was found to destroy malignant melanoma cells in culture under an acidified pH and sufficient concentration through caspase-3 mediated apoptosis. Furthermore, cis-UCA is able to considerably diminish the growth rate in human melanoma tumors on living SCID mice. Using replication-competent Semliki Forest viruses, human melanoma tumors grown in SCID mice were dramatically shrunken as the fulminant production of viruses in melanoma cells leads them to apoptosis within 72 hours. Small oligopeptides attaching to melanoma cells were identified using in vivo phage display. The melanoma-specific peptides found were further tested in vitro on adenoviruses. Ultimately, the adenoviral retargeting using the peptides was tested in vivo. One peptide homed to human transferring receptor upregulated on melanoma cells. In order to kill the malignant melanoma cells with the retargeted adenoviruses, the viruses should carry genetic material producing apoptotic proteins in the cancer tissue. TIMP-3 has been identified as a good candidate for such a protein, as it inhibits malignant cell adhesion as well as promotes apoptosis through a caspase-8 pathway. It is further shown here that adenovirally delivered TIMP-3 is even more potent, as it could kill non-adherent cancer cells, lacking the fully functional death receptor signalling pathway. Adenovirally delivered TIMP-2 also showed marked antitumor effects in human malignant melanoma xenografts on SCID mice both in ex vivo and systemic delivery.

Imaging of Dopamine and Serotonin Transporters. Pre-clinical Studies with Radiotracers for Positron Emission Tomography

The action of the neurotransmitters dopamine (DA) and serotonin (5-HT) at synapses is terminated by their rapid reuptake into presynaptic nerve endings via plasma membrane dopamine (DAT) and serotonin (SERT) transporters. Alterations in the function of these transporters have been suggested as a feature of several neurological and neuropsychiatric diseases, such as Parkinson’s disease (PD), depression, and anxiety. A suitable clinical method for studying these transporters non-invasively in vivo is positron emission tomography (PET) utilizing radiopharmaceuticals (tracers) labelled with short-lived positron-emitting radionuclides. The aim of this study was to evaluate in rats two novel radiotracers, [¹⁸F]beta -CFT-FP and ¹⁸FFMe-McN, for imaging DAT and SERT, respectively, using in vitro, ex vivo and in vivo methods. Substituting an N-methyl in [¹⁸F]beta-CFT, a well known DAT tracer, with a ¹⁸Ffluoropropyl group significantly changed the properties of the tracer. [¹⁸F]beta- CFT showed slow kinetics and metabolism, and a high specific uptake in the striatum, whereas [¹⁸F]beta-CFT-FP showed fast kinetics and metabolism, and a moderate specific uptake in the striatum. [¹⁸F]betaCFT-FP was selective for DAT; but [¹⁸F]beta-CFT also bound to the noradrenaline transporter. [¹⁸F]beta-CFT-FP may be a suitable PET tracer for imaging the striatal DAT sites, but a tracer with a higher affinity is needed for imaging extrastriatal DAT sites. In rats, ¹⁸FFMe-McN showed high target-to-non-target ratios, specificity and selectivity for SERT, but slow kinetics. However, ¹⁸FFMe-McN reveals potential for imaging SERT, at least in pre-clinical studies. In addition, the sensitivities of [¹⁸F]beta CFT and [¹⁸ F]FDOPA (a precursor of DA) for detecting mild nigrostriatal hypofunction were compared in an animal model of PD. The uptake of [¹⁸F]FDOPA was significantly affected by compensatory effects in dopaminergic cells, whereas [¹⁸F]beta-CFT was more sensitive and therefore more suitable for PET studies of mild dopaminergic symptoms. In conclusion, both novel tracers, [¹⁸F]-CFT-FP and ¹⁸FFMe-McN, have potential, but are not optimal PET tracers for DAT and SERT imaging in rats, respectively. [¹⁸F]beta-CFT is superior to [18F]FDOPA for imaging mild nigral lesions in rat brains.

Detection of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of Novel PET Imaging Probes With Experimental Models

Atherosclerosis is a life-long vascular inflammatory disease and the leading cause of death in Finland and in other western societies. The development of atherosclerotic plaques is progressive and they form when lipids begin to accumulate in the vessel wall. This accumulation triggers the migration of inflammatory cells that is a hallmark of vascular inflammation. Often, this plaque will become unstable and form vulnerable plaque which may rupture causing thrombosis and in the worst case, causing myocardial infarction or stroke. Identification of these vulnerable plaques before they rupture could save lives. At present, in the clinic, there exists no appropriated, non-invasive method for their identification. The aim of this thesis was to evaluate novel positron emission tomography (PET) probes for the detection of vulnerable atherosclerotic plaques and to characterize, two mouse models of atherosclerosis. These studies were performed by using ex vivo and in vivo imaging modalities. The vulnerability of atherosclerotic plaques was evaluated as expression of active inflammatory cells, namely macrophages. Age and the duration of high-fat diet had a drastic impact on the development of atherosclerotic plaques in mice. In imaging of atherosclerosis, 6-month-old mice, kept on high-fat diet for 4 months, showed matured, metabolically active, atherosclerotic plaques. [18F]FDG and 68Ga were accumulated in the areas representative of vulnerable plaques. However, the slow clearance of 68Ga limits its use for the plaque imaging. The novel synthesized [68Ga]DOTA-RGD and [18F]EF5 tracers demonstrated efficient uptake in plaques as compared to the healthy vessel wall, but the pharmacokinetic properties of these tracers were not optimal in used models. In conclusion, these studies resulted in the identification of new strategies for the assessment of plaque stability and mouse models of atherosclerosis which could be used for plaque imaging. In the used probe panel, [18F]FDG was the best tracer for plaque imaging. However, further studies are warranted to clarify the applicability of [18F]EF5 and [68Ga]DOTA-RGD for imaging of atherosclerosis with other experimental models.

Equipamento brasileiro de detecção gama intra-operatória para biópsia de linfonodo sentinela

OBJETIVO: Testar a eficácia de equipamento de detecção gama intra-operatória (DGI) desenvolvido pelo IPEN (Brasil), em procedimentos de biópsia de linfonodo sentinela (BLS) no melanoma e no câncer de mama. MÉTODOS: Foram estudados 40 pacientes portadores de melanoma ou câncer de mama com indicação para realização de BLS.Todos pacientes foram submetidos à linfocintilografia e a BLS ocorreu entre 2 a 24 horas após a mesma. Concomitantemente à DGI, realizou-se o mapeamento linfático com corante vital. Foram feitas leituras com o equipamento convencional Neoprobe® 1500 e com o equipamento em teste (IPEN) dos valores de captação do sítio de injeção do radiofármaco, do LS in vivo e ex vivo e da captação de fundo. Foi registrado se o LS estava corado e se o cirurgião teve facilidade para encontrá-lo. Nos primeiros 20 pacientes utilizou-se o equipamento convencional e depois o de teste; nos outros 20, utilizou-se primeiro o equipamento em teste, com objetivo de verificar se o mesmo identificava primariamente o LS. RESULTADOS: Dos quarenta pacientes, 33 eram portadores de tumor de mama e sete de melanoma cutâneo; variação da idade: 21 a 68 anos (mediana= 46 anos); 35 mulheres e 5 homens. Em apenas um paciente o LS não foi encontrado, nem pela DGI nem pelo corante vital. Não houve diferença estatística entre as razões ex vivo/fundo obtidas com os dois equipamentos (p=0, 2583-ns). CONCLUSÃO: É possível realizar o procedimento de BLS com o equipamento brasileiro desenvolvido pelo IPEN, com facilidade e sem prejuízo para o paciente.

Repair of segmental bone defects with fiber-reinforced composite: a study of material development and an animal model on rabbits

The Repair of segmental defects in load-bearing long bones is a challenging task because of the diversity of the load affecting the area; axial, bending, shearing and torsional forces all come together to test the stability/integrity of the bone. The natural biomechanical requirements for bone restorative materials include strength to withstand heavy loads, and adaptivity to conform into a biological environment without disturbing or damaging it. Fiber-reinforced composite (FRC) materials have shown promise, as metals and ceramics have been too rigid, and polymers alone are lacking in strength which is needed for restoration. The versatility of the fiber-reinforced composites also allows tailoring of the composite to meet the multitude of bone properties in the skeleton. The attachment and incorporation of a bone substitute to bone has been advanced by different surface modification methods. Most often this is achieved by the creation of surface texture, which allows bone growth, onto the substitute, creating a mechanical interlocking. Another method is to alter the chemical properties of the surface to create bonding with the bone – for example with a hydroxyapatite (HA) or a bioactive glass (BG) coating. A novel fiber-reinforced composite implant material with a porous surface was developed for bone substitution purposes in load-bearing applications. The material’s biomechanical properties were tailored with unidirectional fiber reinforcement to match the strength of cortical bone. To advance bone growth onto the material, an optimal surface porosity was created by a dissolution process, and an addition of bioactive glass to the material was explored. The effects of dissolution and orientation of the fiber reinforcement were also evaluated for bone-bonding purposes. The Biological response to the implant material was evaluated in a cell culture study to assure the safety of the materials combined. To test the material’s properties in a clinical setting, an animal model was used. A critical-size bone defect in a rabbit’s tibia was used to test the material in a load-bearing application, with short- and long-term follow-up, and a histological evaluation of the incorporation to the host bone. The biomechanical results of the study showed that the material is durable and the tailoring of the properties can be reproduced reliably. The Biological response - ex vivo - to the created surface structure favours the attachment and growth of bone cells, with the additional benefit of bioactive glass appearing on the surface. No toxic reactions to possible agents leaching from the material could be detected in the cell culture study when compared to a nontoxic control material. The mechanical interlocking was enhanced - as expected - with the porosity, whereas the reinforcing fibers protruding from the surface of the implant gave additional strength when tested in a bone-bonding model. Animal experiments verified that the material is capable of withstanding load-bearing conditions in prolonged use without breaking of the material or creating stress shielding effects to the host bone. A Histological examination verified the enhanced incorporation to host bone with an abundance of bone growth onto and over the material. This was achieved with minimal tissue reactions to a foreign body. An FRC implant with surface porosity displays potential in the field of reconstructive surgery, especially regarding large bone defects with high demands on strength and shape retention in load-bearing areas or flat bones such as facial / cranial bones. The benefits of modifying the strength of the material and adjusting the surface properties with fiber reinforcement and bone-bonding additives to meet the requirements of different bone qualities are still to be fully discovered.