64CuCl2 PET Imaging of 4T1-Related Allograft of Triple-Negative Breast Cancer in Mice.

64CuCl2 is an economic radiotracer for oncologic PET investigations. In the present study, we characterized the uptake of 64CuCl2 in vivo by µPET/CT in an allograft 4T1-related mouse model (BALB/c) of advanced breast cancer. 18F-FDG was used as a comparator. Twenty-two animals were imaged 7-9 days following 4T1-cell implantation inside mammary glands. Dynamic 64CuCl2 µPET/CT acquisition or iterative static images up to 8 h p.i. were performed. Animal biodistribution and tumor uptake were first evaluated in vivo by µPET analysis and then assessed on tissue specimens. Concerning 18F-FDG µPET, a static acquisition was performed at 15 min and 60 min p.i. Tumor 64CuCl2 accumulation increased from 5 min to 4 h p.i., reaching a maximum value of 5.0 ± 0.20 %ID/g. Liver, brain, and muscle 64CuCl2 accumulation was stable over time. The tumor-to-muscle ratio remained stable from 1 to 8 h p.i., ranging from 3.0 to 3.7. Ex vivo data were consistent with in vivo estimations. The 18F-FDG tumor accumulation was 8.82 ± 1.03 %ID/g, and the tumor-to-muscle ratio was 4.54 ± 1.11. 64CuCl2 PET/CT provides good characterization of the 4T1-related breast cancer model and allows for exploration of non-glycolytic cellular pathways potentially of interest for theragnostic strategies.

Hypoxia-driven heterogeneous expression of α5 integrin in glioblastoma stem cells is linked to HIF-2α.

Despite numerous molecular targeted therapies tested in glioblastoma (GBM), no significant progress in patient survival has been achieved in the last 20 years in the overall population of GBM patients except with TTfield setup associated with the standard of care chemoradiotherapy. Therapy resistance is associated with target expression heterogeneity and plasticity between tumors and in tumor niches. We focused on α5 integrin implicated in aggressive GBM in preclinical and clinical samples. To address the characteristics of α5 integrin heterogeneity we started with patient data indicating that elevated levels of its mRNA are related to hypoxia pathways. We turned on glioma stem cells which are considered at the apex of tumor formation and recurrence but also as they localize in hypoxic niches. We demonstrated that α5 integrin expression is stem cell line dependent and is modulated positively by hypoxia in vitro. Importantly, heterogeneity of expression is conserved in in vivo stem cell-derived mice xenografts. In hypoxic niches, HIF-2α is preferentially implicated in α5 integrin expression which confers migratory capacity to GBM stem cells. Hence combining HIF-2α and α5 integrin inhibitors resulted in proliferation and migration impairment of α5 integrin expressing cells. Stabilization of HIF-2α is however not sufficient to control integrin α5 expression. Our results show that AHR (aryl hydrocarbon receptor) expression is inversely related to HIF-2α and α5 integrin expressions suggesting a functional competition between the two transcription factors. Collectively, data confirm the high heterogeneity of a GBM therapeutic target, its induction in hypoxic niches by HIF-2α and suggest a new way to attack molecularly defined GBM stem cells.

Improving Seizure Frequency Documentation and Classification.

Background and Objectives: Accurate and reliable seizure data are essential for evaluating treatment strategies and tracking the quality of care in epilepsy clinics. This quality improvement project aimed to increase seizure documentation (i.e., documentation of seizure frequency from 80% to 100%, date of last seizure from 35% to 50%, and International League Against Epilepsy (ILAE) seizure classification from 35% to at least 50%) over 6 months. Methods: We surveyed 7 epileptologists to determine their perceived seizure frequency, ILAE classification, and date of last seizure documentation habits. Baseline data were collected weekly from September to December 2021. Subsequently, we implemented a newly created flowsheet in our Electronic Health Record (EHR) based on the Epilepsy Learning Healthcare System (ELHS) Case Report Forms to increase seizure documentation in a standardized way. Two epileptologists tested this flowsheet tool in their epilepsy clinics between February 2022 and July 2022. Data were collected weekly and compared with documentation from other epileptologists within the same group. Results: Epileptologists at our center believed they documented seizure frequency for 84%-87% of clinic visits, which aligned with baseline data collection, showing they recorded seizure frequency for 83% of clinic visits. Epileptologists believed they documented ILAE classification for 47%-52% of clinic visits, and baseline data showed this was documented in 33% of clinic visits. They also reported documenting the date of the last seizure for 52%-63% of clinic visits, but this occurred in only 35% of clinic visits. After implementing the new flowsheet, documentation increased to nearly 100% for all fields being completed by the providers who tested the flowsheet. Discussion: We demonstrated that by implementing an easy-to-use standardized EHR documentation tool, our documentation of critical metrics, as defined by the ELHS, improved dramatically. This shows that simple and practical interventions can substantially improve clinically meaningful documentation.

Performance evaluation of the IRIS XL-220 PET/CT system, a new camera dedicated to non-human primates.

BACKGROUND: Non-human primates (NHP) are critical in biomedical research to better understand the pathophysiology of diseases and develop new therapies. Based on its translational and longitudinal abilities along with its non-invasiveness, PET/CT systems dedicated to non-human primates can play an important role for future discoveries in medical research. The aim of this study was to evaluate the performance of a new PET/CT system dedicated to NHP imaging, the IRIS XL-220 developed by Inviscan SAS. This was performed based on the National Electrical Manufacturers Association (NEMA) NU 4-2008 standard recommendations (NEMA) to characterize the spatial resolution, the scatter fraction, the sensitivity, the count rate, and the image quality of the system. Besides, the system was evaluated in real conditions with two NHP with 18F-FDG and (-)-[18F]FEOBV which targets the vesicular acetylcholine transporter, and one rat using 18F-FDG. RESULTS: The full width at half maximum obtained with the 3D OSEM algorithm ranged between 0.89 and 2.11 mm in the field of view. Maximum sensitivity in the 400-620 keV and 250-750 keV energy windows were 2.37% (22 cps/kBq) and 2.81% (25 cps/kBq), respectively. The maximum noise equivalent count rate (NEC) for a rat phantom was 82 kcps at 75 MBq and 88 kcps at 75 MBq for energy window of 250-750 and 400-620 keV, respectively. For the monkey phantom, the maximum NEC was 18 kcps at 126 MBq and 19 kcps at 126 MBq for energy window of 250-750 and 400-620 keV, respectively. The IRIS XL provided an excellent quality of images in non-human primates and rats using 18F-FDG. The images acquired using (-)-[18F]FEOBV were consistent with those previously reported in non-human primates. CONCLUSIONS: Taken together, these results showed that the IRIS XL-220 is a high-resolution system well suited for PET/CT imaging in non-human primates.

Role of the RNA-binding protein HuR in human renal cell carcinoma.

Human conventional renal cell carcinoma (CRCC) remains resistant to therapy. The RNA-binding protein HuR regulates the stability and/or translation of multiple messenger RNAs involved in malignant transformation. In this study, we aimed to evaluate the potential role of HuR in this pathology. Using seven human CRCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as 15 normal/renal cell carcinoma tumor pairs, we showed that HuR is overexpressed in all tumors independently of the VHL status. Futhermore, HuR cytoplasmic presence appears to be more common in early tumor stages, suggesting a role in tumor promotion. We then assessed the effect of HuR knockdown using small interfering RNA in cultured cell and in tumor-bearing mice. Both in vitro and in vivo, we observed that cell growth was inhibited by 60% and that this effect was obtained through an inhibition of cell proliferation and an induction of cell apoptosis. Finally, we found that expression of vascular endothelium growth factor, tumor growth factor-beta and of the hypoxia-induced transcription factor-2alpha as well as the constitutive activation of the oncogenic phosphoinositide 3-kinase/Akt, nuclear factor-kappaB and mitogen-activated protein kinase pathways were decreased in HuR-depleted cells and tumors. All these results suggest a pivotal role for HuR in human CRCC.

Nutrition knowledge of rural older populations: can congregate meal site participants manage their own diets?

Congregate meal sites were funded to assist socioeconomically disadvantaged, rural older individuals in improving their health-related practices. Although the participants in the program are largely female, the meals are designed to meet one third of the daily caloric intake of a 70-year-old male, and to satisfy his recommended dietary allowances for total fat, fiber, calcium, and sodium. The actual percentage of the required nutrient intake contributed by meals served at congregate sites is indefinite. Moreover, the ability of congregate meal participants to manage their diets and their receptiveness to helpful nutrition information in that regard is unknown. Our objective was to promote nutritional knowledge in economically disadvantaged, rural older participants by studying its impact on their ability to benefit from congregate meal programs. We used a test, intervention, retest methodology to examine the effect of short-term nutrition interventions on congregate meal site participants' nutrition knowledge. The objective was to determine the participants' potential for managing their own diets (e.g., their ability to determine what diet behaviors are appropriate for specific chronic conditions). We found that while congregate meal site participants have knowledge of nutrition recommendations, their ability to apply this information in helping themselves to prevent or control their chronic conditions remains in question.

von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR.

We have shown that parathyroid hormone-related protein (PTHrP) is a survival factor for human renal cell carcinoma (RCC) and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of messenger RNA (mRNA) stability, as observed for tumor growth factors (TGFs). Our goals were to analyze the alternative splicing of PTHrP mRNA in human RCC and from these results to identify VHL/hypoxia-induced factor (HIF) system-regulated mRNA-binding proteins involved in PTHrP mRNA stability. We used: (i) a panel of human RCC cells expressing or not VHL; (ii) VHL-deficient 786-0 cells transfected with active or inactive VHL and (iii) human RCC samples and corresponding normal tissues. By quantitative real-time reverse transcription-polymerase chain reaction analysis, the 141 PTHrP mRNA isoform was found to be predominant in all cells and tumors (80%). In cells transfected with VHL, the expressions of all isoforms were decreased by 50%. Eight proteins with molecular weights ranging from 20 to 75 kDa were found to bind to biotinylated transcripts spanning the 141 PTHrP mRNA AU-rich 3'-untranslated region whose abundancy was dependent on VHL expression. The protein having an apparent molecular weight of 30 kDa was identified by western blot as HuR, a RNA-binding protein with stabilizing functions on various mRNA coding for proteins important in malignant transformation including vascular endothelial growth factor and TGF-beta. PTHrP expression studies confirmed the involvement of HuR in PTHrP upregulation in this disease. Common mRNA-binding proteins regulated by the VHL/HIF system may constitute new therapeutic opportunities against human RCC that remains refractory to therapies.

The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth.

BACKGROUND: Human clear cell renal cell carcinoma (CRCC) remains resistant to therapies. Recent advances in Hypoxia Inducible Factors (HIF) molecular network led to targeted therapies, but unfortunately with only limited clinical significance. Elucidating the molecular processes involved in kidney tumorigenesis and resistance is central to the development of improved therapies, not only for kidney cancer but for many, if not all, cancer types. The oncogenic PI3K/Akt, NF-kB and MAPK pathways are critical for tumorigenesis. The sonic hedgehog (SHH) signaling pathway is crucial to normal development. RESULTS: By quantitative RT-PCR and immunoblot, we report that the SHH signaling pathway is constitutively reactivated in tumors independently of the von Hippel-Lindau (VHL) tumor suppressor gene expression which is inactivated in the majority of CRCC. The inhibition of the SHH signaling pathway by the specific inhibitor cyclopamine abolished CRCC cell growth as assessed by cell counting, BrdU incorporation studies, fluorescence-activated cell sorting and beta-galactosidase staining. Importantly, inhibition of the SHH pathway induced tumor regression in nude mice through inhibition of cell proliferation and neo-vascularization, and induction of apoptosis but not senescence assessed by in vivo studies, immunoblot and immunohistochemistry. Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-beta were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition. Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition. CONCLUSIONS: These findings support targeting SHH for the treatment of CRCC and pave the way for innovative and additional investigations in a broad range of cancers.

Synthesis of a Novel Electrospun Polycaprolactone Scaffold Functionalized with Ibuprofen for Periodontal Regeneration: An In Vitro andIn Vivo Study.

Ibuprofen (IBU) has been shown to improve periodontal treatment outcomes. The aim of this study was to develop a new anti-inflammatory scaffold by functionalizing an electrospun nanofibrous poly-ε-caprolactone membrane with IBU (IBU-PCL) and to evaluate its impact on periodontal inflammation, wound healing and regeneration in vitro and in vivo. IBU-PCL was synthesized through electrospinning. The effects of IBU-PCL on the proliferation and migration of epithelial cells (EC) and fibroblasts (FB) exposed to Porphyromonas gingivlais lipopolysaccharide (Pg-LPS) were evaluated through the AlamarBlue test and scratch assay, respectively. Anti-inflammatory and remodeling properties were investigated through Real time qPCR. Finally, the in vivo efficacy of the IBU-PCL membrane was assessed in an experimental periodontitis mouse model through histomorphometric analysis. The results showed that the anti-inflammatory effects of IBU on gingival cells were effectively amplified using the functionalized membrane. IBU-PCL reduced the proliferation and migration of cells challenged by Pg-LPS, as well as the expression of fibronectin-1, collagen-IV, integrin α3ß1 and laminin-5. In vivo, the membranes significantly improved the clinical attachment and IBU-PCL also reduced inflammation-induced bone destruction. These data showed that the IBU-PCL membrane could efficiently and differentially control inflammatory and migratory gingival cell responses and potentially promote periodontal regeneration.

Imaging thrombosis with 99mTc-labeled RAM.1-antibody in vivo.

INTRODUCTION: Platelets play a major role in thrombo-embolic diseases, notably by forming a thrombus that can ultimately occlude a vessel. This may provoke ischemic pathologies such as myocardial infarction, stroke or peripheral artery diseases, which represent the major causes of death worldwide. The aim of this study was to evaluate the specificity of radiolabeled Rat-Anti-Mouse antibody (RAM.1). METHODS: We describe a method to detect platelets by using a RAM.1 coupled with the chelating agent hydrazinonicotinic acid (HYNIC) conjugated to 99mTc, for Single Photon Emission Computed Tomography (SPECT). To induce platelet accumulation at a site of interest, we used a mouse model of FeCl3 induced injury of the carotid artery. 90 min after i.v. injection of [99mTc][Tc(HYNIC)-RAM.1], biodistribution of the radiolabeled RAM.1 was assessed, SPECT imaging and histological analysis were performed on the mice that underwent FeCl3-induced vessel damage. RESULTS: We demonstrated a quick and strong affinity of the radiolabeled RAM.1 for the platelet thrombus. Results clearly demonstrated the ability of this radioimmunoconjugate for detecting thrombi from 10 min post injection with an exceptional thrombi uptake. Using FeCl3, the median ratio between the thrombus and the background was 12.4 (range 9.3-42.3) as compared to 1.0 (range: 0.86-2.7) p < 0.05 when using 0.9% NaCl. CONCLUSION: Thanks to the high sensitivity of SPECT, we provided evidence that [99mTc][Tc(HYNIC)-RAM.1] represents a powerful tool to detect localized platelet thrombi which could potentially be used in humans. Because of the relative low cost and high sensitivity, these results encourage further study like the detection of non-induced thrombus and further developments toward clinical application. This is further supported by the fact that RAM.1 recognizes human platelets.

Cardiovascular and survival effects of sympatho-inhibitors in adriamycin-induced cardiomyopathy in rats.

Adriamycin (ADR) is a widely used drug for the treatments of cancers. This study evaluates the effects of moxonidine and metoprolol on cardiac hemodynamics and survival in ADR-induced left ventricular dysfunction (total dose of 20 mg/kg in a 4-week regimen). Rats were treated with the centrally acting I(1)R agonist sympatho-inhibitor, moxonidine, or with the non-selective beta-adrenergic antagonist, metoprolol, during 1 month or until death. Treatments began 1 week after the onset of the ADR administration. Low doses (0.5 and 1 mg/kg/day) of moxonidine and metoprolol (10 mg/kg/day) improved cardiovascular function. High doses of moxonidine (3 mg/kg/day) and metoprolol (150 mg/kg/day) were cardiodepressive. Moxonidine and metoprolol both failed to improve survival. These data indicate that a treatment with these sympatho-inhibitors can reduce the left ventricular dysfunction induced by ADR. Moreover, these cardioprotective effects where obtained even when ADR was used at a dose regimen usually employed for its antineoplastic effects in rodents. Nevertheless, in this particular cardiomyopathy, we did not find any association between improvements of functional parameters and survival whatever the drug and the dose used. This problem points out the difficulty to prevent, at least with sympatho-inhibitory drugs alone, the mortality linked to the chronic cardiotoxicity of ADR.

Hemodynamic and cardiac anti-hypertrophic actions of clonidine in Goldblatt one-kidney, one-clip rats.

In congestive heart failure, the chronic sympathetic hyperactivity contributes to a poor prognosis. In this respect, clonidine, a centrally acting sympathoinhibitory drug, has previously been tested in clinical trials. The aim of the current study was to evaluate the effects of clonidine on morbidity and mortality in an experimental model of cardiac hypertrophy associated with hypertension, renal failure, and intense sympathetic activation. One-kidney, one-clip Goldblatt hypertensive rats were treated orally with clonidine (200 microg/kg/d) during 30 days and were compared with untreated rats and with sham-operated animals. Cardiac hemodynamics, left ventricular volume and elasticity, cardiac morphometry and histology, and renal function were evaluated. A survival study was also performed. Clonidine normalized cardiac function, ventricular stiffness, and prevented ventricular structural remodeling. Moreover, despite a marked renal function impairment, survival of the animals was increased in the clonidine group. The centrally acting sympathoinhibitory drug clonidine exhibited marked cardioprotective properties. This study emphasized the interest of evaluating drugs whose aim is to treat congestive heart failure, in an experimental model of cardiac and renal failure.

Chronic treatment with rilmenidine in spontaneously hypertensive rats: differences between two schedules of administration.

Rilmenidine is one of the lead compounds of the second generation of centrally acting antihypertensive drugs. In the first part of this study, 2 routes of administration of chronic treatment (1 month) with rilmenidine were compared. In conscious and pentobarbital-anesthetized spontaneously hypertensive rats (SHR), rilmenidine was delivered intraperitoneally either 250 microg/kg b.i.d. or 500 microg/kg/d infusion by means of minipumps. The possibility of rilmenidine-induced desensitization of central (brain cortex) and/or peripheral (kidney) alpha2-adrenoreceptors was studied in saturation experiments with the classic alpha2-adrenergic antagonist [H]rauwolscine. In the second part of this study, the cardiovascular and cardiac antihypertrophic effects of the most efficient procedure were investigated. The discontinuous administration of the drug was more effective than infusion. In rats treated with rilmenidine b.i.d., mean blood pressure was reduced by nearly 15% when no reduction occurred in SHRs treated with minipumps. With the first schedule of administration, plasma concentration of the drug reached a maximum of approximately 30 ng/ml when it was only 12 ng/ml with the continuous infusion of the same dose. Anesthesia with pentobarbital potentiated the antihypertensive effect of rilmenidine in rats treated discontinuously and unmasked an antihypertensive action in rats receiving the drug with minipumps. In saturation binding experiments, no significant changes in adrenergic receptors were observed in kidney membrane preparations. In contrast, in brain cortical membranes a reduction by about 50% of the Bmax of [H]rauwolscine value was observed in rats treated discontinuously with rilmenidine. In contrast, a 400% increase of the Bmax was observed in the brain of rats treated with minipumps. Over the one-month period of the second study, the discontinuous treatment with the 500 microg/kg/d dose of rilmenidine was still able to reduce blood pressure, at least at the peak concentration time, but did not induce any significant reduction of the ventricular mass. In conclusion, rilmenidine has only weak antihypertensive effects in conscious SHRs, even at doses higher than those that are active in rabbits and humans. As a consequence, it lacks significant cardiac antihypertrophic effects in this species. Pharmacokinetic data show that the rapid plasma withdrawal of this drug may explain this particular feature in rats.