Papers by Alessandra Pistilli
PubMed, Jul 1, 1996
Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tail... more Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.
Frontiers in Endocrinology
IntroductionAmong substances released into the environment by anthropogenic activities, the heavy... more IntroductionAmong substances released into the environment by anthropogenic activities, the heavy metal cadmium (Cd) is known to induce severe testicular injury causing male subfertility/infertility. Zinc (Zn) is another heavy metal that, unlike Cd, is physiologically present in the testis, being essential for spermatogenesis. We aimed to examine the possibility that 50 µM ZnCl2 could counteract the toxic effects induced by Cd in an in vitro model of porcine prepubertal Sertoli cells (SCs) exposed to both subtoxic (5 μM) and toxic (10 μM) concentrations of CdCl2 for 48 h.Materials and MethodsApoptosis, cell cycle, and cell functionality were assessed. The gene expression of Nrf2 and its downstream antioxidant enzymes, ERK1/2, and AKT kinase signaling pathways were evaluated.Materials and ResultsWe found that Zn, in co-treatment with subtoxic and toxic Cd concentration, increased the number of metabolically active SCs compared to Cd exposure alone but restored SC functionality only i...
Toxicology in Vitro, 2018
Long-term exposure to cigarette smoke induces severe injuries to respiratory system through sever... more Long-term exposure to cigarette smoke induces severe injuries to respiratory system through several mechanisms, some of them are well defined, but many others are not yet elucidated. Beside its classical role in nervous system, we have previously shown that Nerve Growth Factor (NGF) and its receptors have a crucial role in airway inflammatory diseases, such as Chronic Obstructive Pulmonary Disease. To expand our knowledge about the relevance of NGF and its receptors in airway diseases induced by cigarette smoking, we exposed for 16 weeks the bronchial epithelial cell line BEAS-2B to sub-toxic concentrations of whole cigarette smoke extract or pure nicotine. Viability, cell cycle gene expression, cell morphology and migration ability were tested and compared to NGF release and gene expression. Modulation of its receptors TrKA and p75NTR was also analyzed. The present study shows that long term exposure of BEAS-2B cells to cigarette smoke extract or nicotine induces: (A) differences: in cell viability, in the expression of cell cycle-related genes, in NGF release and in gene expression of NGF and its receptors; (B) similarities: in morphology and migration ability. Taken together, our data provide new insights about the biological role of NGF and its receptors in airway diseases induced by long-term cigarette smoking and, finally, our data evidence the opportunity not to use nicotine lozenges or e-cigarettes as anti smoking replacement therapy in patients with a previous airway disease according to the ability of nicotine to increase the amount of the pro-inflammatory cytokine NGF into the bronchial environment.
International Journal of Environmental Research and Public Health
Environmental tobacco smoke remains a major risk factor, for both smokers and non-smokers, able t... more Environmental tobacco smoke remains a major risk factor, for both smokers and non-smokers, able to trigger the initiation and/or the progression of several human diseases. Although in recent times governments have acted with the aim of banning or strongly reducing its impact within public places and common spaces, environmental tobacco smoke remains a major pollutant in private places, such as the home environment or cars. Several inflammatory and long-term biomarkers have been analysed and well-described, but the list of mediators modulated during the early phases of inhalation of environmental tobacco smoke needs to be expanded. The aim of this study was to measure the short-term effects after exposure to side-stream smoke on Nerve Growth Factor and its receptors Tropomyosin-related kinase A and neurotrophin p75, molecules already described in health conditions and respiratory diseases. Twenty-one non-smokers were exposed to a home-standardized level of SS as well as to control sm...
Free Radical Biology and Medicine
Frontiers in Urology
The aim of our study was to investigate the plasma NGF concentration and TrkA/p75NTR receptor exp... more The aim of our study was to investigate the plasma NGF concentration and TrkA/p75NTR receptor expression on white blood cells (WBCs), in peripheral and corpus cavernosum blood isolated from patients with erectile dysfunction and metabolic syndrome (ED/MetS). This was a pilot case–control study. Inclusion criteria were as follows: men 18–65 years with ED and MetS and healthy subjects. The first sampling was performed at the level of the cubital vein (VC). Subsequently, 20 μg of intracavernous alprostadil was administered, and a second blood draw from the corpora cavernosa (CC) was performed once erection was achieved. Subsequently, the third blood sample was repeated at the level of the VC. We enrolled 8 cases with ED/MetS and 8 controls. There was no significant difference between the case and control group in terms of mean age (49.3 ± 5.9 and 53.13 ± 8.9, respectively). The case group had a lower IIEF score compared to the control group (14 ± 3.2 versus 27.3 ± 2.1; p < 0.05). De...
IUBMB Life, 2021
Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-h... more Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1αmediated transcriptional regulation of stress response genes with antiinflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.
Administration of prohibited substances to enhance athletic performance represents an emerging me... more Administration of prohibited substances to enhance athletic performance represents an emerging medical, social, ethical and legal issue. Traditional controls are based on direct detection of substances or their catabolites. However out-of-competition doping may not be easily revealed by standard analytical methods. Alternative indirect control strategies are based on the evaluation of mid- and long-term effects of doping in tissues. Drug-induced long-lasting changes of gene expression may be taken as effective indicators of doping exposure. To validate this approach, we used real-time PCR to monitor the expression pattern of selected genes in human haematopoietic cells exposed to nandrolone, insulin-like growth factor I (IGF-I) or growth hormone (GH). Some candidate genes were found significantly and consistently modulated by treatments. Nandrolone up-regulated AR, ESR2 and PGR in K562 cells, and SRD5A1, PPARA and JAK2 in Jurkat cells; IGF-I up-regulated EPOR and PGR in HL60 cells, ...
Theriogenology, 2020
The influence of NGF in male reproduction in some animal species and humans has already been asse... more The influence of NGF in male reproduction in some animal species and humans has already been assessed. Many of these effects are mediated by the distribution and abundance of tropomyosin receptor kinase A (TrKA) and p75 neurotrophin (p75NTR) receptors on sperm cells. The aim of this research was to investigate the role of NGF and its receptors, TrKA and p75NTR, in rabbit sperm outcomes during in vitro storage. Major semen traits (kinetic parameters, apoptotic, necrotic and live sperm) were recorded in rabbit semen samples from 0 to 12 h of storage (every 4 h). Three experimental hypotheses were formulated: i) sperm storage changes NGF receptor abundance in rabbit sperm; ii) TrKA and p75NTR differently modulate NGF signalling (assessed by the neutralisation of receptors); iii) NGF-receptor interactions show different responses during storage (evaluated by the addition of exogenous NGF). The results demonstrate that: (i) the receptor number changed in a time-dependent manner with a significant increase in p75NTR after 8e12 h of storage; ii) the neutralisation of NGF receptors largely affected VCL, apoptotic, necrotic and live cells during sperm storage, i.e. blockade of TrKA significantly increased speed, capacitation, necrosis and apoptosis, whereas blockade of p75NTR improved motility and live cells; iii) the addition of exogenous human NGF (100 ng/mL) at different time points of storage (0, 4, 8 h) differently influenced sperm traits i.e. NGF addition at time 0 positively affected all the pro-vital traits (kinetic, live cells) whereas, after 4e8 h, the effect of NGF was null or negative. In conclusion, NGF affects kinetic and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm in a timedependent manner. Most of these modifications are modulated by the receptors involved (TrKA or p75NTR), which changed considerably during sperm storage (increase of p75NTR).
Journal of Vascular Surgery: Venous and Lymphatic Disorders, 2018
Background: The aim of this systematic review was to provide comprehensive data on the prevalence... more Background: The aim of this systematic review was to provide comprehensive data on the prevalence of variations of the saphenofemoral junction (SFJ) to prevent misidentification of the SFJ or the incomplete ligation of the tributaries of the great saphenous vein. Methods: A systematic review was conducted using the PubMed, Embase, and Cochrane Library databases through September 14, 2017. To be included in the meta-analysis, a study had to report prevalence data on the morphology of the SFJ or the presence of venous tributaries. Results: A total of 16 studies (7433 legs) were included. The majority of studies were performed during varicose vein surgery (74.14%), with fewer studies by means of computed tomography venography and cadaveric dissection. The pooled prevalence estimate (PPE) for a duplication of the SFJ with a bifid junction was 9.6% (P ¼ .001). The PPE for a duplication of the SFJ with two separate junctions was 1.7%. The PPE for ectasia of the SFJ was 2.3% in type 1, 1.2% in type 2, and 1.7% in type 3. The distribution of the PPE for the number of venous SFJ tributaries was approximately normal with a slight right skew; a higher rate was observed in the group with four venous tributaries to the SFJ. Conclusions: This analysis found high heterogeneity in the prevalence of SFJ anatomic variants and the number of venous SFJ tributaries. For this reason, it is highly recommended that a preoperative Doppler ultrasound assessment of the SFJ and great saphenous vein be performed.
Frontiers in physiology, 2018
Advanced aging, vascular dysfunction, and nitric oxide (NO) bioavailability are recognized risk f... more Advanced aging, vascular dysfunction, and nitric oxide (NO) bioavailability are recognized risk factors for Alzheimer's disease (AD). However, the contribution of AD, , to this putative pathophysiological mechanism is still unclear. To better answer this point, we quantified cortical perfusion with arterial spin labeling (PVC-CBF), measured ultrasound internal carotid (ICA), and femoral (FA) artery blood flow in a group of patients with similar age (~78 years) but different cognitive impairment (i.e., mild cognitive impairment MCI, mild AD-AD1, moderate AD-AD2, and severe AD-AD3) and compared them to young and healthy old (aged-matched) controls. NO-metabolites and passive leg-movement (PLM) induced hyperemia were used to assess systemic vascular function. Ninety-eight individuals were recruited for this study. PVC-CBF, ICA, and FA blood flow were markedly (range of 9-17%) and significantly (all < 0.05) reduced across the spectrum from YG to OLD, MCI, AD1, AD2, AD3 subjects. ...
European Journal of Nutrition, 2017
Purpose Human melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistan... more Purpose Human melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown. Methods Three different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment. Results We demonstrated that SFN strongly decreased cell viability and proliferation, induced G 2 /M cell cycle arrest, promoted apoptosis through the activation of caspases 3, 8, 9 and hampered migration and invasion abilities in the melanoma cell lines. Remarkably, HEMa cells were not affected by SFN treatment. Transcriptomic analysis revealed regulation of genes involved in response to stress, apoptosis/cell death and metabolic processes. SFN upregulated the expression of pro-apoptotic genes, such as p53, BAX, PUMA, FAS and MDM2; promoted cell cycle inhibition and growth arrest by upregulating EGR1, GADD45B, ATF3 and CDKN1A; and simultaneously acted as a potent inhibitor of genotoxicity by launching the stress-inducible protein network (HMOX1, HSPA1A, HSPA6, SOD1). Conclusion Overall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent.
Biological Chemistry, 2017
Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous ... more Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous studies have shown the involvement of β-NGF and its receptors TrKA and p75NTR in OA-related pain, but their role in its pathogenesis is still unclear. The aim of our study was to investigate the amount of β-NGF and the expression levels of its receptors on cells isolated from synovial fluid and blood from OA patients who had undergone total knee arthroplasty, in order to check any possible correlation with the disease staging. Our results show a progressive stage-related increase of β-NGF and its receptors both in serum and synovial fluid. Furthermore, with respect to control subjects, OA patients show an increased amount of inflammatory monocytes along with an increased expression of β-NGF, TrKA and p75NTR. In conclusion, our study suggests a stage-related modulation of β-NGF and its receptors in the inflammatory process of OA.
Veterinary Anaesthesia and Analgesia, 2017
Objective To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro.... more Objective To assess the effects of xylazine and dexmedetomidine on equine chondrocytes, in vitro. Study design Prospective, experimental study. Study material Equine articular chondrocytes from five male horses.
Journal of Alzheimer's Disease, 2016
Italian journal of anatomy and embryology, Nov 11, 2010
Italian journal of anatomy and embryology, 2011
Reproduction in domestic animals = Zuchthygiene, 2015
Experiments were devised to characterize the expression of nerve growth factor, beta polypeptide ... more Experiments were devised to characterize the expression of nerve growth factor, beta polypeptide (NGF), and its cognate receptors neurotrophic tyrosine kinase receptor type 1 (NTRK1) and nerve growth factor receptor (NGFR) in rabbit male sex organs, as well as the concentrations of NGF in both seminal and blood plasma of sexually mature male rabbits. Immunoreactivity and gene expression for NGF and cognate receptors were detected in testis, prostate gland and seminal vesicle. The highest levels of NGF and NTRK1 transcripts were found in the prostate, while intermediate expressions were found in the testis. NGFR transcripts were expressed at the same levels in both testis and prostate and were more abundant than in seminal vesicles. The widespread distribution of NGF in all prostate glandular cells, together with its relative high mRNA abundance, confirms that the prostate of rabbits is the main source of this neurotrophin. In conclusion, the present data suggest that the NGF system ...
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Papers by Alessandra Pistilli