Rapid Etiological Classification of Meningitis by NMR Spectroscopy Based on Metabolite Profiles and Host Response

Bacterial meningitis is an acute disease with high mortality that is reduced by early treatment. Identification of the causative microorganism by culture is sensitive but slow. Large volumes of cerebrospinal fluid (CSF) are required to maximise sensitivity and establish a provisional diagnosis. We have utilised nuclear magnetic resonance (NMR) spectroscopy to rapidly characterise the biochemical profile of CSF from normal rats and animals with pneumococcal or cryptococcal meningitis. Use of a miniaturised capillary NMR system overcame limitations caused by small CSF volumes and low metabolite concentrations. The analysis of the complex NMR spectroscopic data by a supervised statistical classification strategy included major, minor and unidentified metabolites. Reproducible spectral profiles were generated within less than three minutes, and revealed differences in the relative amounts of glucose, lactate, citrate, amino acid residues, acetate and polyols in the three groups. Contributions from microbial metabolism and inflammatory cells were evident. The computerised statistical classification strategy is based on both major metabolites and minor, partially unidentified metabolites. This data analysis proved highly specific for diagnosis (100% specificity in the final validation set), provided those with visible blood contamination were excluded from analysis; 6-8% of samples were classified as indeterminate. This proof of principle study suggests that a rapid etiologic diagnosis of meningitis is possible without prior culture. The method can be fully automated and avoids delays due to processing and selective identification of specific pathogens that are inherent in DNA-based techniques

The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction

Rationale: Pluripotent stem cells (PSCs) are being investigated as a cell source for regenerative medicine since they provide an infinitive pool of cells that are able to differentiate towards every cell type of the body. One possible therapeutic application involves the use of these cells to treat myocardial infarction (MI), a condition where billions of cardiomyocytes (CMs) are lost. Although several protocols have been developed to differentiate PSCs towards CMs, none of these provide a completely pure population, thereby still posing a risk for neoplastic teratoma formation. Therefore, we developed a strategy to (i) monitor cell behavior noninvasively via site-specific integration of firefly luciferase (Fluc) and the human positron emission tomography (PET) imaging reporter genes, sodium iodide symporter (hNIS) and somatostatin receptor type 2 (hSSTr2), and (ii) perform hSSTr2-mediated suicide gene therapy via the clinically used radiopharmacon 177Lu-DOTATATE. Methods: Human embryonic stem cells (ESCs) were gene-edited via zinc finger nucleases to express Fluc and either hNIS or hSSTr2 in the safe harbor locus, adeno-associated virus integration site 1. Firstly, these cells were exposed to 4.8 MBq 177Lu-DOTATATE in vitro and cell survival was monitored via bioluminescence imaging (BLI). Afterwards, hNIS+ and hSSTr2+ ESCs were transplanted subcutaneously and teratomas were allowed to form. At day 59, baseline 124I and 68Ga-DOTATATE PET and BLI scans were performed. The day after, animals received either saline or 55 MBq 177Lu-DOTATATE. Weekly BLI scans were performed, accompanied by 124I and 68Ga-DOTATATE PET scans at days 87 and 88, respectively. Finally, hSSTr2+ ESCs were differentiated towards CMs and transplanted intramyocardially in the border zone of an infarct that was induced by left anterior descending coronary artery ligation. After transplantation, the animals were monitored via BLI and PET, while global cardiac function was evaluated using cardiac magnetic resonance imaging. Results: Teratoma growth of both hNIS+ and hSSTr2+ ESCs could be followed noninvasively over time by both PET and BLI. After 177Lu-DOTATATE administration, successful cell killing of the hSSTr2+ ESCs was achieved both in vitro and in vivo, indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2+ ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed.Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment

In Vivo Tracking of Human Neural Stem Cells with 19F Magnetic Resonance Imaging

(c)The Author

High fat diet treatment impairs hippocampal long-term potentiation without alterations of the core neuropathological features of Alzheimer disease

Type 2 diabetes (T2DM) and obesity might increase the risk for AD by 2-fold. Different attempts to model the effect of diet-induced diabetes on AD pathology in transgenic animal models, resulted in opposite conclusions. Here, we used a novel knock-in mouse model for AD, which, differently from other models, does not overexpress any proteins. Long-term high fat diet treatment triggers a reduction in hippocampal N-acetyl-aspartate/myo-inositol metabolites ratio and impairs long term potentiation in hippocampal acute slices. Interestingly, these alterations do not correlate with changes in the core neuropathological features of AD, i.e. amyloidosis and Tau hyperphosphorylation. The data suggest that AD phenotypes associated with high fat diet treatment seen in other models for AD might be exacerbated because of the overexpressing systems used to study the effects of familial AD mutations. Our work supports the increasing insight that knock-in mice might be more relevant models to study the link between metabolic disorders and AD

Low-Dose Imaging in a New Preclinical Total-Body PET/CT Scanner.

Ionizing radiation constitutes a health risk to imaging scientists and study animals. Both PET and CT produce ionizing radiation. CT doses in pre-clinical in vivo imaging typically range from 50 to 1,000 mGy and biological effects in mice at this dose range have been previously described. [ <sup>18</sup> F]FDG body doses in mice have been estimated to be in the range of 100 mGy for [ <sup>18</sup> F]FDG. Yearly, the average whole body doses due to handling of activity by PET technologists are reported to be 3-8 mSv. A preclinical PET/CT system is presented with design features which make it suitable for small animal low-dose imaging. The CT subsystem uses a X-source power that is optimized for small animal imaging. The system design incorporates a spatial beam shaper coupled with a highly sensitive flat-panel detector and very fast acquisition (<10 s) which allows for whole body scans with doses as low as 3 mGy. The mouse total-body PET subsystem uses a detector architecture based on continuous crystals, coupled to SiPM arrays and a readout based in rows and columns. The PET field of view is 150 mm axial and 80 mm transaxial. The high solid-angle coverage of the sample and the use of continuous crystals achieve a sensitivity of 9% (NEMA) that can be leveraged for use of low tracer doses and/or performing rapid scans. The low-dose imaging capabilities of the total-body PET subsystem were tested with NEMA phantoms, in tumor models, a mouse bone metabolism scan and a rat heart dynamic scan. The CT imaging capabilities were tested in mice and in a low contrast phantom. The PET low-dose phantom and animal experiments provide evidence that image quality suitable for preclinical PET studies is achieved. Furthermore, CT image contrast using low dose scan settings was suitable as a reference for PET scans. Total-body mouse PET/CT studies could be completed with total doses of <10 mGy

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer's disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes

Mechanisms of vasomotor sympathetic activity generation in the renovascular hypertension in Wistar rats

The renovascular arterial hypertension (AH) is associated with sympathetic hyperactivity and impaired baroreflex sensitivity determined by increase in circulating angiotensin II (Ang II). The aim of this thesis was to evaluate the role of oxidative stress in the generation and maintenance of the AH (2 Kidney – 1 Clip). Five series of experiments were performed in male Wistar. The results showed autonomic cardiovascular control changes characterized by increased sympathetic modulation in blood vessels, impaired baroreflex sensitivity and tachycardia from the fourth week. Furthermore, we observed that Ang II (via AT-1 receptor) and NO (produced by iNOS) in the rostral ventrolateral medulla (RVLM) are involved in the maintenance of AH and renal sympathetic nerve activity in the 2K-1C. Indeed, there is an imbalance between pro-oxidants and antioxidants in the RVL and hypothalamic paraventricular nucleus (PVN) of animals 2K-1C, because the increase in blood pressure (BP) and sympathetic hyperactivity was reversed in response to administration of the antioxidant (Tempol) into the RVLM and PVN. The over expression of CuZnSOD in the RVLM prevented the AH and reduced the production of superoxide in this region in 2K-1C. Furthermore, improved the impaired baroreflex sensitivity and inhibited the tachycardia observed from the fourth week of AH. Chronic Vitamin C treatment decreased BP and sympathetictactivation in hypertensive animals. In addition, reduced gene expression of subunits of NAD (P) H oxidase in the PVN and RVLM, but unchanged the expression of CuZnSOD. Oxidative stress also seems to influence the gene expression of AT-1 receptor, iNOS, nNOS, IL-1 â and Glut-1 in the PVN and RVL regions, because the treatment reduced the expression of these components. Based on our results, we suggest that the angiotensin system, NO and oxidative stress in the PVN and RVLM are involved in the maintenance of sympathetic tone and the AH 2K-1C. Oxidative stress in sympathetic premotor neurons is a major mechanism for the establishment and maintenance of Ang II – dependent hypertension.A Hipertensão arterial (HA) renovascular está associada com uma hiperatividade simpática e prejuízo na sensibilidade reflexa do barorreceptor determinadas por aumento na Angiotensina II (Ang II) circulante. O objetivo da presente Tese foi avaliar o papel do estresse oxidativo na geração e manutenção da HA 2 Rins-1Clipe. Ratos Machos Wistar foram utilizados em cinco séries de experimentos. Os resultados demonstraram uma alteração no controle autonômico cardíaco caracterizado por maior modulação simpática nos vasos, um prejuízo da sensibilidade reflexa do barorreceptor e taquicardia a partir da quarta semana. Além disso, observou-se que Ang II (via receptor AT-1) e o NO (produzido pela iNOS) na região rostro ventrolateral do bulbo (RVL), participam mantendo a HA e a atividade nervosa simpática renal em ratos 2R-1C. Evidenciou-se um desequilíbrio entre próoxidantes e antioxidantes, caracterizando um estado de estresse oxidativo nas regiões RVL e no núcleo paraventricular (PVN) dos animais 2R-1C, visto que o aumento de pressão arterial (PA) e a hiperatividade simpática são revertidos com a administração do antioxidante (Tempol) nessas regiões. A super expressão crônica de CuZnSOD na região RVL preveniu a HA e reduziu a produção de superóxidos nessa região em ratos 2R-1C. Além disso, melhorou a sensibilidade reflexa do barorreceptor e inibiu a taquicardia observada a partir da quarta semana de HA. O tratamento crônico com Vitamina C promoveu queda da PA e simpato-inibição nos animais hipertensos. Além disso, reduziu a expressão gênica das subunidades de NAD(P)H oxidase nas regiões RVL e PVN, mas não interferiu na expressão de CuZnSOD. O estresse oxidativo também parece influenciar na expressão gênica do receptor AT-1, iNOS, nNOS, IL-1 ƒÀ e Glut-1 nas regioes RVL e PVN, visto que o tratamento reduziu a expressao desses componentes. Baseado em nossos resultados, podemos sugerir que o sistema angiotensinergico, nitrergico e o estresse oxidativo nas regioes RVL e PVN estao envolvidos na manutencao do tonus simpatico e da HA 2R-1C. O estresse oxidativo em neuronios pre-motores do simpatico e um mecanismo essencial para o estabelecimento e manutencao da HA Ang II - dependente.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP: 05/60151-6CAPES/BEX: 3496/07-4TEDEBV UNIFESP: Teses e dissertaçõe

Enoxaparin for symptomatic COVID-19 managed in the ambulatory setting: An individual patient level analysis of the OVID and ETHIC trials.

BACKGROUND: Antithrombotic treatment may improve the disease course in non-critically ill, symptomatic COVID-19 outpatients. METHODS: We performed an individual patient-level analysis of the OVID and ETHIC randomized controlled trials, which compared enoxaparin thromboprophylaxis for either 14 (OVID) or 21 days (ETHIC) vs. no thromboprophylaxis for outpatients with symptomatic COVID-19 and at least one additional risk factor. The primary efficacy outcome included all-cause hospitalization and all-cause death within 30 days from randomization. Both studies were prematurely stopped for futility. Secondary efficacy outcomes were major symptomatic venous thromboembolic events, arterial cardiovascular events, or their composite occurring within 30 days from randomization. The same outcomes were assessed over a 90-day follow-up. The primary safety outcome was major bleeding (ISTH criteria). RESULTS: A total of 691 patients were randomized: 339 to receive enoxaparin and 352 to the control group. Over 30-day follow-up, the primary efficacy outcome occurred in 6.0 % of patients in the enoxaparin group vs. 5.8 % of controls for a risk ratio (RR) of 1.05 (95%CI 0.57-1.92). The incidence of major symptomatic venous thromboembolic events and arterial cardiovascular events was 0.9 % vs. 1.8 %, respectively (RR 0.52; 95%CI 0.13-2.06). Most cardiovascular thromboembolic events were represented by symptomatic venous thromboembolic events, occurring in 0.6 % vs. 1.5 % of patients, respectively. A similar distribution of outcomes between the treatment groups was observed over 90 days. No major bleeding occurred in the enoxaparin group vs. one (0.3 %) in the control group. CONCLUSIONS: We found no evidence for the clinical benefit of early administration of enoxaparin thromboprophylaxis in outpatients with symptomatic COVID-19. These results should be interpreted taking into consideration the relatively low occurrence of events

Search for the decay J/ψ→γ+invisibleJ/\psi\to\gamma + \rm {invisible}

We search for $J/\psi$ radiative decays into a weakly interacting neutral particle, namely an invisible particle, using the $J/\psi$ produced through the process $\psi(3686)\to\pi^+\pi^-J/\psi$ in a data sample of $(448.1\pm2.9)\times 10^6$ $\psi(3686)$ decays collected by the BESIII detector at BEPCII. No significant signal is observed. Using a modified frequentist method, upper limits on the branching fractions are set under different assumptions of invisible particle masses up to 1.2 $\mathrm{\ Ge\kern -0.1em V}/c^2$. The upper limit corresponding to an invisible particle with zero mass is 7.0$\times 10^{-7}$ at the 90\% confidence level