BODIPY-Based Analogue of the TREM2-Binding Molecular Adjuvant Sulfavant A, a Chemical Tool for Imaging and Tracking Biological Systems.

Recently, we described synthetic sulfolipids named Sulfavants as a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. The members of this family, Sulfavant A (1), Sulfavant R (2), and Sulfavant S (3), showed important effects on triggering receptor expressed on myeloid cells 2 (TREM2)-induced differentiation and maturation of human dendritic cells (hDC), through a novel cell mechanism underlying the regulation of the immune response. As these molecules are involved in biological TREM2-mediated processes crucial for cell survival, here, we report the synthesis and application of a fluorescent analogue of Sulfavant A bearing the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene moiety (Me4-BODIPY). The fluorescent derivative, named PB-SULF A (4), preserving the biological activity of Sulfavants, opens the way to chemical biology and cell biology experiments to better understand the interactions with cellular and in vivo organ targets and to improve our comprehension of complex molecular mechanisms underlying the not fully understood ligand-induced TREM2 activity.

High-resolution, handheld camera use for occult breast lesion localization plus sentinel node biopsy (SNOLL): a single-institution experience with 186 patients.

BACKGROUND: Sentinel node and occult lesion localization (SNOLL) calls for a combination of two specific procedures: intraoperative detection of sentinel lymph node (SLN) via gamma probe and radioguided occult lesion localization (ROLL). This applies to nonpalpable invasive breast cancer or high-grade in situ carcinoma. As opposed to standard techniques, today's handheld gamma cameras enable intraoperative scintigraphic images. METHODS: A cohort (N = 186) of consecutive patients with breast cancer was subjected to radioguided conservative surgery (quadrantectomy and SLN biopsy), using a standard gamma probe and a high-resolution handheld camera. Intraoperative SLN frozen section was also performed. RESULTS: Neoplastic lesions were removed in 99.4% of all patients, and SLN biopsy was achieved in 99%. Of the 137 patients with invasive cancer, SLN metastasis was confirmed in 21. In 12% of patients, a second operation was required for close or tumor-positive surgical margins. DISCUSSION: This combination of procedures represents an improvement in the surgical management of occult breast carcinomas and is the method of choice for accurate tumor localization and SLN biopsy. Handheld cameras have the potential to become highly useful intraoperative aids.

FMR1 deletion in rats induces hyperactivity with no changes in striatal dopamine transporter availability.

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder emerging in early life characterized by impairments in social interaction, poor verbal and non-verbal communication, and repetitive patterns of behaviors. Among the best-known genetic risk factors for ASD, there are mutations causing the loss of the Fragile X Messenger Ribonucleoprotein 1 (FMRP) leading to Fragile X syndrome (FXS), a common form of inherited intellectual disability and the leading monogenic cause of ASD. Being a pivotal regulator of motor activity, motivation, attention, and reward processing, dopaminergic neurotransmission has a key role in several neuropsychiatric disorders, including ASD. Fmr1 Δexon 8 rats have been validated as a genetic model of ASD based on FMR1 deletion, and they are also a rat model of FXS. Here, we performed behavioral, biochemical and in vivo SPECT neuroimaging experiments to investigate whether Fmr1 Δexon 8 rats display ASD-like repetitive behaviors associated with changes in striatal dopamine transporter (DAT) availability assessed through in vivo SPECT neuroimaging. At the behavioral level, Fmr1 Δexon 8 rats displayed hyperactivity in the open field test in the absence of repetitive behaviors in the hole board test. However, these behavioral alterations were not associated with changes in striatal DAT availability as assessed by non-invasive in vivo SPECT and Western blot analyses.

Role of Nuclear Imaging to Understand the Neural Substrates of Brain Disorders in Laboratory Animals: Current Status and Future Prospects.

Molecular imaging, which allows the real-time visualization, characterization and measurement of biological processes, is becoming increasingly used in neuroscience research. Scintigraphy techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) provide qualitative and quantitative measurement of brain activity in both physiological and pathological states. Laboratory animals, and rodents in particular, are essential in neuroscience research, providing plenty of models of brain disorders. The development of innovative high-resolution small animal imaging systems together with their radiotracers pave the way to the study of brain functioning and neurotransmitter release during behavioral tasks in rodents. The assessment of local changes in the release of neurotransmitters associated with the performance of a given behavioral task is a turning point for the development of new potential drugs for psychiatric and neurological disorders. This review addresses the role of SPECT and PET small animal imaging systems for a better understanding of brain functioning in health and disease states. Brain imaging in rodent models faces a series of challenges since it acts within the boundaries of current imaging in terms of sensitivity and spatial resolution. Several topics are discussed, including technical considerations regarding the strengths and weaknesses of both technologies. Moreover, the application of some of the radioligands developed for small animal nuclear imaging studies is discussed. Then, we examine the changes in metabolic and neurotransmitter activity in various brain areas during task-induced neural activation with special regard to the imaging of opioid, dopaminergic and cannabinoid receptors. Finally, we discuss the current status providing future perspectives on the most innovative imaging techniques in small laboratory animals. The challenges and solutions discussed here might be useful to better understand brain functioning allowing the translation of preclinical results into clinical applications.

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea.

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6-/- COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1-/- with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods.

High-resolution, hand-held camera for sentinel-node detection.

BACKGROUND: The imaging probe (IP) is a high-resolution (HR), 1-in(2) field-of-view hand-held gamma camera. We used it to detect breast cancer sentinel node (SN). PATIENTS AND METHODS: We divided 120 T1 breast cancer patients, who underwent Anger camera lymphoscintigraphy (ACL), in two subgroups of 60 patients who were age, body mass index, and cancer size matched: subgroup A (SA) and B (SB). SN was detected with a common gamma probe (GP) in SA, with IP plus GP in SB. RESULTS: Surgeons removed radioactive nodes without exceeding four nodes. Eighty-two (82) SNs were taken off in SA and 105 in SB (p<0.01). Of SA, 22 of 60 patients and 36 of 60 patients of SB showed more than 1 node, and 3 of them showed 3 nodes and 1 showed 4 nodes. Thirteen (13) patients resulted N(+) (21.6%) in SA. Ten (10) patients of SA showed an invasion on the hottest nodes and 3 on the second nodes. In the SB, 18 patients (25%) showed invasion. Sixteen (16) invasions were on hot, 4 on second, and 1 on the third node. Withdrawal time of SN was 11.25+/-4.7 minutes for SA and 7.4+/-2.8 minutes for SB (p<0.025). CONCLUSIONS: SN biopsy with IP is fast and discovers more SNs and more invasions than ACL.

Directional probe for radio-guided surgery: A pilot study.

PURPOSE: The sentinel lymph node (SLN) biopsy technique has highly evolved during the last 20 yr. Consequently, the intraoperative use of Gamma Probes (GPs) for SLN mapping is increased. This preliminary study evaluates a novel directional GP prototype. This proof-of-concept prototype is designed to identify the direction of radiopharmaceuticals uptakes, by combining the information from multiple detectors. The purpose of this work is to develop a tool able to effectively guide the surgeon reducing the surgery time. METHODS: The proposed prototype consists of three CsI(Tl) scintillation crystals, each coupled with an S10931 silicon photomultiplier (Hamamatsu Photonics K.K., Hamamatsu, JP). The three detectors lie on the same plane with an angle of 30° between them. The central detector is placed as in a common GP, so it can be used to pinpoint the target tissue. Meanwhile, the lateral sensors provide a broader view of the surgical field. A dedicated data acquisition system digitizes and processes the signals from the front-end electronics. Finally, an embedded system, based on ARM processor, calculates and displays the acquired count rates. In order to assess the prototype behavior, the isosensitivity curves for the three detectors were measured. Meanwhile, for the central one, the main quality criteria measurements were also performed (i.e., sensitivity, radial sensitivity, and spatial resolution). RESULTS: For the central detector, the measured sensitivity at the tip of the probe is better than 5 cps/kBq. The full width at half maximum (FWHM) of the radial sensitivity is less than 30° and the FWHM of the lateral sensitivity (spatial resolution) is about 7.2 mm. The central detector measured isosensitivity distribution shows a narrow profile in agreement with the spatial resolution measured. On the contrary, the two lateral detectors exhibit widespread isosensitivity distributions that mean a larger field of view. The system had shown satisfactory performance and reliability, meeting the minimal requirements of gamma probe systems. CONCLUSIONS: The prototype presented in this paper allows a rapid localization by the use of the whole system, while the sole central detector can be used to pinpoint the target source. This device, unlike common GPs, allows localizing simultaneously different areas of radiopharmaceuticals uptake, thus precisely guiding the surgeon to the region of interest. These preliminary results encourage to develop a further prototype for intraoperative validation.

Thyroid cancer imaging in vivo by targeting the anti-apoptotic molecule galectin-3.

BACKGROUND: The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. METHODS: The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific (99m)Tc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 microCi of (99m)Tc-labeled mAb to galectin-3 (30 microg protein/in 100 microl saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. FINDINGS: Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. CONCLUSIONS: These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed.