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During development and in various diseases of the CNS, new blood vessel formation starts with endothelial tip cell selection and vascular sprout migration, followed by the establishment of functional, perfused blood vessels. Here we describe a method that allows the assessment of these distinct angiogenic steps together with antibody-based protein detection in the postnatal mouse brain. Intravascular and perivascular markers such as Evans blue (EB), isolectin B4 (IB4) or laminin (LN) are used alongside simultaneous immunofluorescence on the same sections. By using confocal laser-scanning microscopy and stereological methods for analysis, detailed quantification of the 3D postnatal brain vasculature for perfused and nonperfused vessels (e.g., vascular volume fraction, vessel length and number, number of branch points and perfusion status of the newly formed vessels) and characterization of sprouting activity (e.g., endothelial tip cell density, filopodia number) can be obtained. The entire protocol, from mouse perfusion to vessel analysis, takes ∼10 d.

Purpose: Most hyperprogression disease (HPD) definitions are based on tumor growth rate (TGR). However, there is still no consensus on how to evaluate this phenomenon. Experimental Design: We investigated two independent cohorts of patients with advanced solid tumors treated in phase I trials with (i) programmed cell death 1 (PD-1)/PD-L1 antibodies in monotherapy or combination and (ii) targeted agents (TA) in unapproved indications. A Response Evaluation Criteria in Solid Tumors (RECIST) 1.1-based definition of hyperprogression was developed. The primary endpoint was the assessment of the rate of HPD in patients treated with ICIs or TAs using both criteria (RECIST and TGR) and the impact on overall survival (OS) in patients who achieved PD as best response. Results: Among 270 evaluable patients treated with PD-1/PD-L1 inhibitors, 29 PD-1/PD-L1-treated patients (10.7%) had HPD by RECIST definition. This group had a significantly lower OS (median of 5.23 months; 95% CI, 3.97-6.45) when compared with the non-HPD progressor group (median, 7.33 months; 95% CI, 4.53-10.12; HR ¼ 1.73, 95% CI, 1.05-2.85; P ¼ 0.04). In a subset of 221 evaluable patients, 14 (6.3%) were categorized as HPD using TGR criteria, differences in median OS (mOS) between this group (mOS 4.2 months; 95% IC, 2.07-6.33) and non-HPD progressors (n ¼ 44) by TGR criteria (mOS 6.27 months; 95% CI, 3.88-8.67) were not statistically significant (HR 1.4, 95% IC, 0.70-2.77; P ¼ 0.346). Among 239 evaluable patients treated with TAs, 26 (10.9%) were classified as having HPD by RECIST and 14 using TGR criteria in a subset of patients. No differences in OS were observed between HPD and non-HPD progressors treated with TAs. Conclusions: HPD measured by TGR or by RECIST was observed in both cohorts of patients; however, in our series, there was an impact on survival only in the immune-checkpoint inhibitor cohort when evaluated by RECIST. We propose a new way to capture HPD using RECIST criteria that is intuitive and easy to use in daily clinical practice.

Expression of neurotrophins (NTs) and their receptors is elevated in the adult CNS under several neuropathological conditions. We have investigated the anatomical and electrophysiological consequences of chronic NT-3 or NT-4/5 treatment on established organotypic hippocampal slice cultures maintained in vitro for > 14 days. Both NT-3 and NT-4/5 increased spontaneous, action potential-dependent excitatory synaptic activity (sEPSCs), but only NT-3 increased inhibitory synaptic activity (sIPSCs) in CA3 pyramidal cells. Both NTs strongly promoted spontaneous synaptic bursting activity. Spontaneous bursts of EPSCs were observed after either NT treatment but only NT-3-treated cultures exhibited an increase in spontaneous bursts of IPSCs. In addition, sIPSC bursts were eliminated by blocking glutamatergic excitation. The frequency of miniature inhibitory postsynaptic currents, but not miniature excitatory postsynaptic currents, was also increased by both NT-3 and NT-4/5. Furthermore, NT-3 and NT-4/5 induced an up-regulation of the growth-associated protein GAP-43, suggesting that neurotrophins may be able to induce axonal reorganization in established neuronal networks. CA1 pyramidal cells exhibited slight alterations in dendritic branching after NT-4/5, but not NT-3 treatment. We conclude that chronic treatment with NT-3 or NT-4/5 can affect an established hippocampal network by elevating spontaneous inhibitory and excitatory synaptic activity and inducing coordinated pre- and postsynaptic structural changes.

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM–1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered lowmolecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/ megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp:: 1 / 2 vdbp-mCherry) zebrafish line expressing in the liver the Nterminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (1 / 2 vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.

Background information. Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O 2) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca 2+ concentration, mitochondrial free-Ca 2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcriptionpolymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. Results. Hypoxia induced an increase in intracellular and mitochondrial free-Ca 2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH 2-terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. Conclusions and Significance. Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

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