Immunosurveillance of the liver by intravascular effector CD8(+) T cells.

Effector CD8(+) T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.

Spiro[chromane-2,4'-piperidine]-based histone deacetylase inhibitors with improved in vivo activity.

A series of spiro[chromane-2,4'-piperidine] derivatives based on a previously published lead benzyl spirocycle 1 and bearing various N-aryl and N-alkylaryl substituents on the piperidine ring were prepared as novel histone deacetylase (HDAC) inhibitors. The compounds were evaluated for their abilities to inhibit nuclear HDACs, their in vitro antiproliferative activities, and in vitro ADME profiles. Based on these activities, 4-fluorobenzyl and 2-phenylethyl spirocycles were selected for further characterization. In vivo pharmacokinetic (PK) studies showed that both compounds exhibit an overall lower clearance rate, an increased half-life, and higher AUCs after intravenous and oral administration than spiropiperidine 1 under the conditions used. The improved PK behavior of these two compounds also correlated with superior in vivo antitumor activity in an HCT-116 xenograft model.

Angiogenesis inhibition by the maleimide-based small molecule GNX-686.

We investigated the anti-angiogenic properties of GNX-686, a newly identified maleimide-based small molecule. In vitro studies on HUVEC showed that GNX-686 inhibited cell growth with an ED(50) of 20-25 µM, while human HeLa tumor cells and non-transformed embryonic mouse fibroblasts were less sensitive for the drug. More importantly, at 4 µM, a concentration that was non-toxic to any cell in culture, GNX-686 showed a significant inhibitory effect on tube formation by HUVEC, indicating a profound anti-angiogenic activity. Angiogenesis inhibition was subsequenly tested in the chorioallantoic membrane (CAM) of the chicken embryo. A significant angiostatic activity was observed in the CAM model, and results were compared with the effect of bevacizumab, a well known and clinically used VEGF inhibitor. Under our experimental conditions, GNX-686 was found to be as effective as bevacizumab, significantly changing the morphology of the vascular network, as illustrated and quantified by the relative number of branching points and the relative mean mesh size of the vascular network. In another in vivo model of neovascularization, the mouse retinopathy of prematurity (ROP), the vascular network of GNX-686-treated mice was significantly altered, reducing the density of the retinal microvasculature, as compared to the control retinas. Immunohistochemical processing of the GNX-686 treated (4µM) eyes showed over 50% reduction of the number of cell nuclei associated with neovasculature, as compared to the control-treated eye. Taken together these results demonstrate that GNX-686 is a promising anti-angiogenic compound that could be developed for the treatment of diseases characterized by aberrant angiogenesis such as ocular pathologies and cancer.

Discovery, synthesis, and pharmacological evaluation of spiropiperidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors.

New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.

Synthesis and biological evaluation of N-hydroxyphenylacrylamides and N-hydroxypyridin-2-ylacrylamides as novel histone deacetylase inhibitors.

The histone deacetylases (HDACs) are able to regulate gene expression, and histone deacetylase inhibitors (HDACi) emerged as a new class of agents in the treatment of cancer as well as other human disorders such as neurodegenerative diseases. In the present investigation, we report on the synthesis and biological evaluation of compounds derived from the expansion of a HDAC inhibitor scaffold having N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-3-(pyridin-2-yl)-2-propenamide as core structures and containing a phenyloxopropenyl moiety, either unsubstituted or substituted by a 4-methylpiperazin-1-yl or 4-methylpiperazin-1-ylmethyl group. The compounds were evaluated for their ability to inhibit nuclear HDACs, as well as for their in vitro antiproliferative activity. Moreover, their metabolic stability in microsomes and aqueous solubility were studied and selected compounds were further characterized by in vivo pharmacokinetic experiments. These compounds showed a remarkable stability in vivo, compared to hydroxamic acid HDAC inhibitors that have already entered clinical trials. The representative compound 30b showed in vivo antitumor activity in a human colon carcinoma xenograft model.

Septal grafts restore cognitive abilities and amyloid precursor protein metabolism.

Cortical cholinergic loss and amyloidogenic processing of the beta-amyloid precursor protein (APP), may functionally interact in Alzheimer's disease. However, it is still unknown whether biological restoration of regulatory cholinergic inputs affects APP metabolism in vivo. Rats immunolesioned with 192 IgG-saporin exhibited severe acquisition deficits in place navigation that were paralleled by a dramatic loss of terminal cholinergic innervation and by marked changes in the regional expression of APP-like immunoreactivity. Moreover, in these animals, we observed a drastic reduction of soluble APP (sAPP) and a concomitant increase of the unsoluble, membrane-bound fraction (mAPP). Notably, at about 6 months post-surgery, lesioned animals implanted with reinnervating cholinergic-rich septal tissue grafts exhibited fairly normal spatial navigation abilities, as well as cortical and hippocampal APP levels that were restored up to normal or near-normal values. APP levels correlated significantly with lesion- or graft-induced changes in cholinergic innervation density, and both these measures correlated with performance in the spatial navigation task. Thus, integrity of ascending cholinergic inputs may be required to prevent amyloidogenic processing of APP in vivo and to modulate cognitive performance.

Acetylcholine release from fetal tissue homotopically grafted to the motoneuron-depleted lumbar spinal cord. An in vivo microdialysis study in the awake rat.

Grafts of spinal cord (SC) tissue can survive and develop into the severed SC, but no conclusive data are available concerning the functional activity of transplanted neurons. In the present study, suspensions of prelabeled embryonic ventral SC tissue were grafted to the lumbar SC of rats with motoneuron loss induced by perinatal injection of volkensin. Eight to ten months post-grafting, acetylcholine (ACh) release was measured by microdialysis in awake rats, under either basal or stimulated conditions. In normal animals, baseline ACh output averaged 1.6 pmol/30 microl, it exhibited a 4-fold increase after KCl-induced depolarization or handling, and it was completely inhibited by tetrodotoxin administration. Moreover, ACh levels did not change following acute SC transection performed under anesthesia during ongoing dialysis, suggesting an intrinsic source for spinal ACh. Treatment with volkensin produced a severe (>85%) motoneuronal loss accompanied by a similar reduction in baseline ACh release and almost completely abolished effects of depolarization or handling. In transplanted animals, many motoneuron-like labeled cells were found within and just outside the graft area, but apparently in no case were they able to extend fibers towards the denervated muscle. However, the grafts restored baseline ACh output up to near-normal levels and responded with significantly increased release to depolarization, but not to handling. The present findings indicate that spinal neuroblasts can survive and develop within the motoneuron-depleted SC and release ACh in a near-normal, but apparently non-regulated, manner. This may be of importance for future studies involving intraspinal stem cell grafts.

Neural stem and progenitor cells: choosing the right Shc.

Neural stem cell (NSCs) are self-renewing, multipotent cells able to generate neurons, astrocytes and oligodendrocytes. Since their identification, these properties have made NSCs an attractive subject for therapeutic applications to the damaged brain. In this context, understanding the mechanisms and the molecules regulating their biological properties is important and it is focused to gain control over their proliferative and differentiative potential. Here we will discuss values and unsolved aspects of the system and the employment of potentially key molecular targets for proper control of NSCs fate.

Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.

Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.

Neural stem cells: a pharmacological tool for brain diseases?

Stem cells are believed to provide a tool by which new cells and tissues can be made and by which damaged ones can be replaced or repaired. Over the past few years, the existence of a subset of stem cells has been documented in the fetal brain, therefore named neural stem cells (NSCs). To this regard, the more recent demonstration that similar cells are present in the adult mammalian brain and retain the capability to produce new neurons, has undermined the dogma that neurons are only generated during the fetal life and has stimulated investigations into the regulation and role of adult neurogenesis. Here, we will review the recent advancements on the biology of brain stem cells and discuss the mechanisms and drugs regulating adult neurogenesis, aiming at better estimating the possible future applications of NSCs for brain repair.