Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer.

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1-2) COPD patients compared to severe-very severe (GOLD 3-4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1-2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

Using two dyes to observe the competition of Ca2+ trapping mechanisms and their effect on intracellular Ca2+ signals.

The specificity and universality of intracellular [Formula: see text] signals rely on the variety of spatio-temporal patterns that the [Formula: see text] concentration can display. [Formula: see text] liberation through inositol 1,4,5-trisphosphate receptors ([Formula: see text]) is key for this variety. In this paper, we study how the competition between buffers of different kinetics affects [Formula: see text] signals that involve [Formula: see text] release through [Formula: see text]. The study also provides insight into the underlying spatial distribution of the channels that participate in the signals. Previous works on the effects of [Formula: see text] buffers have drawn conclusions 'indirectly' by observing the [Formula: see text]-bound dye distributions in the presence of varying concentrations of exogenous buffers and using simulations to interpret the results. In this paper, we make visible the invisible by observing the signals simultaneously with two dyes, [Formula: see text] and [Formula: see text], each of which plays the role of a slow or fast [Formula: see text] buffer, respectively. Our observations obtained for different concentrations of [Formula: see text] highlight the dual role that fast buffers exert on the dynamics, either reducing the intracluster channel coupling or preventing channel inhibition and allowing the occurrence of relatively long cycles of [Formula: see text] release. Our experiments also show that signals with relatively high [Formula: see text] release rates remain localized in the presence of large [Formula: see text] concentrations, while the mean speed of the elicited waves increases. We interpret this as a consequence of the more effective uncoupling between [Formula: see text] clusters as the slow dye concentration increases. Combining the analysis of the experiments with numerical simulations, we also conclude that [Formula: see text] release not only occurs within the close vicinity of the centers of the clearly identifiable release sites ([Formula: see text] clusters) but there are also functional [Formula: see text] in between them.

3D geophysical imaging for site-specific characterization plan of an old landfill.

As it is well-known, the characterization plan of an old landfill site is the first stage of the project for the treatment and reclamation of contaminated lands. It is a preliminary in-situ study, with collection of data related to pollution phenomena, and is aimed at defining the physical properties and the geometry of fill materials as well as the possible migration paths of pollutants to the surrounding environmental targets (subsoil and groundwater). To properly evaluate the extent and potential for subsoil contamination, waste volume and possible leachate emissions from the landfill have to be assessed. In such perspective, the integrated use of geophysical methods is an important tool as it allows a detailed 3D representation of the whole system, i.e. waste body and hosting environment (surrounding rocks). This paper presents a very accurate physical and structural characterization of an old landfill and encasing rocks obtained by an integrated analysis of data coming from a multi-methodological geophysical exploration. Moreover, drillings were carried out for waste sampling and characterization of the landfill body, as well as for calibration of the geophysical modeling.

Finite driving rate and anisotropy effects in landslide modeling.

In order to characterize landslide frequency-size distributions and individuate hazard scenarios and their possible precursors, we investigate a cellular automaton where the effects of a finite driving rate and the anisotropy are taken into account. The model is able to reproduce observed features of landslide events, such as power-law distributions, as experimentally reported. We analyze the key role of the driving rate and show that, as it is increased, a crossover from power-law to non-power-law behaviors occurs. Finally, a systematic investigation of the model on varying its anisotropy factors is performed and the full diagram of its dynamical behaviors is presented.

EGF-responsive rat neural stem cells: molecular follow-up of neuron and astrocyte differentiation in vitro.

Neural stem cells (NSCs) could be very useful for the "cell therapy" treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF-responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF-containing medium, cells were plated on poly-L-ornithine-coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the "p53 network," was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.

Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1.

AIMS/HYPOTHESIS: This study aimed to evaluate the effects of hyperglycaemia on the evolution of myocardial infarction and the expression of the transcriptional factor for angiogenesis hypoxia-inducible factor 1alpha (HIF-1alpha) in the rat. METHODS: We studied the effects of streptozotocin induced diabetes on infarct size and HIF-1 alpha gene expression. These parameters were also evaluated in isolated hearts of non-diabetic rat, in condition of high glucose concentration. RESULTS: In streptozotocin (STZ)-diabetic rats (in vivo study), myocardial infarct size was greater (p<0.01) in hyperglycaemic rats (22 mmol/l) than in normoglycaemic (7 mmol/l) or non-diabetic rats. In euglycaemic conditions, basal expression of HIF-1alpha mRNA was not appreciable, but increased steadily after ischaemia (762+/-86%, p<0.001); this response was blunted in hyperglycaemic STZ-rats (6.8+/-6% of the control, p<0.001) and improved in euglycaemic STZ-rats (58+/-10%). The changes in myocardial Rac1 mRNA expression paralleled those of HIF-1alpha. In isolated hearts from non-diabetic rats (in vitro study), perfusion with high glucose (33 mmol/l) produced an infarct size (58+/-2% of the area at risk) not different from that obtained in hyperglycaemic STZ-rats (57+/-2%). Similar changes in the expression of HIF-1alpha and Rac1, which were prevented by glutathione infusion (0.3 mmol/l) were also observed. CONCLUSION/INTERPRETATION: Both hyperglycaemia and high glucose concentrations increased basal HIF-1alpha and Rac1 expression, suggesting a state of pseudohypoxia. These findings show that myocardial infarct size in the rat is increased in hyperglycaemic conditions and is associated with a reduced expression of the HIF-1alpha gene. These changes are reversed, totally or partially, by normoglycaemia or glutathione suggesting a role for reactive oxygen species generation brought about by hyperglycaemia.

RB2/p130 ectopic gene expression in neuroblastoma stem cells: evidence of cell-fate restriction and induction of differentiation.

The activity of the RB2/p130 gene, which is a member of the retinoblastoma gene family, is cell-cycle-regulated and plays a key role in growth inhibition and differentiation. We used neuroblastoma cell lines as a model for studies on neural crest progenitor cell differentiation. We show that Rb2/p130 ectopic protein expression induces morphological and molecular modifications, promoting differentiation of intermediate (I) phenotype SK-N-BE(2)-C neuroblastoma cells towards a neuroblastic (N) rather than a Schwann/glial/melanocytic (S) phenotype. These modifications are stable as they persist even after treatment with an S-phenotype inducer. Rb2/p130 ectopic expression also induces a more differentiated phenotype in N-type SH-SY-5Y cells. Further, this function appears to be independent of cell-cycle withdrawal. The data reported suggest that the Rb2/p130 protein is able to induce neuronal lineage specification and differentiation in neural crest stem and committed neuroblastoma cells, respectively. Thus, the Rb2/p130 protein seems to be required throughout the full neural maturation process.

Endothelin-1 increases cholinergic nerve-mediated contraction of human bronchi via tachykinin synthesis induction.

1. In some asthmatics, muscarinic receptor antagonists are effective in limiting bronchoconstrictor response, suggesting an abnormal cholinergic drive in these subjects. There is a growing body of evidences indicating that cholinergic neurotransmission is also enhanced by endothelin-1 (ET-1) in rabbit bronchi, mouse trachea and in human isolated airway preparations. 2. We investigated the role of secondary mediators in ET-1 induced potentiation of cholinergic nerve-mediated contraction in human bronchi, in particular the possible role of neuropeptides in this phenomenon. 3. Bronchial tissues after endothelin treatment were exposed to a standard electrical field stimulation (EFS) (30% of EFS 30 Hz)-induced contraction. In addition, in some experiments, preparations were treated with a tachykinin NK(2) receptor antagonist and subsequently exposed to the same protocol. HPLC and RIA were performed on organ bath fluid samples. Moreover, the human bronchi were used for the beta-PPT (preprotachykinin) mRNA extraction and semiquantitative reverse transcription polymerase chain reaction (RT - PCR), prior to and 30-40 min following ET-1 challenge. 4. The selective tachykinin NK(2) receptor antagonist, SR48968, was effective to reduce ET-1 potentiation of EFS mediated contraction. HPLC or RIA showed significant increased quantities of NKA in organ bath effluents after EFS stimulation in bronchi pretreated with ET-1. Finally, beta-PPT mRNA level after stimulation of bronchi with ET-1 was increased about 2 fold respect to control untreated bronchi. 5. In conclusion, this study demonstrated that, at least in part, the ET-1 potentiation of cholinergic nerve-mediated contraction is mediated by tachykinin release, suggesting that in addition to nerves, several type of cells, such as airway smooth muscle cell, may participate to neuropeptide production.

pRb2/p130 gene overexpression induces astrocyte differentiation.

There are many data on the activity of the RB gene in neural differentiation and apoptosis, but the role of pRb2/p130 in neuronal and glial maturation has been far less investigated. To elucidate the role of pRb2/p130 in astrocyte development we overexpressed this protein in astrocytoma and normal astrocyte cultures by adenoviral-mediated gene transfer. In astrocytoma cells, p130/RB2 overexpression resulted in a significant reduction of cell growth and in an increased G(0)/G(1) cell population. We did not observe any induction of programmed cell death as determined by TUNEL reaction. Interestingly, pRb2/p130 overexpression induced astrocyte differentiation. Astrocyte cell cycle arrest and differentiation seemed to proceed through a way distinct from the p53 pathway.

In vivo effects of partial phosphorothioated AT1 receptor antisense oligonucleotides in spontaneously hypertensive and normotensive rats.

Partial phosphorothioate (PS) antisense oligodeoxynucleotides (ODNs) targeted against rat AT1 receptor mRNA have been used to control blood pressure in normotensive (WKY) and spontaneously hypertensive (SHR) rats. Molecules were injected intracerebroventricularly (i.c.v., right lateral ventricle) in freely moving animals. The antisense ODN lowered the mean arterial pressure (MAP) 24 hours (-43 mmHg+/-10) and 48 hours (-30 mmHg+/-13) after injection, while the control ODN molecule had no significant effects. The observed decrease of blood pressure was due to a specific inhibition of AT1 receptor gene expression, since the level of its mRNA, monitored by reverse transcription (RT)- polymerase chain reaction (PCR), was significantly reduced by antisense molecule (-40%), compared to sense one. In normotensive rats no effect on MAP have been observed, while AT1 receptor gene expression is reduced (-40%) by antisense treatment. It is known that SHRs have an enhanced basal activity of the central renin-angiotensin system that induces an increase in central sympathetic outflow. Instead in WKY rats the central sympathetic outflow is not conditioned by the enhanced activity of brain renin-angiotensin system. Therefore in normotensive rats although partial PS ODN reduces the AT1 mRNA level this will not result in a modification of the sympathetic outflow and no change in MAP level would be observed.

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc-interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G(0)/G(1) cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell.