PLATELET-ASSOCIATED TISSUE FACTOR EXPRESSION: INSIGHTS INTO THE MEGAKARYOCYTE-PLATELET AXIS.

Tissue factor is the main activator of the blood coagulation cascade and for this reason levels of TF are not easily detectable in cells in contact with blood under physiological conditions. By contrast, in pathological conditions endothelial cells and monocytes can be induced to express TF. Induced-TF is present in atherosclerotic plaques (vessel wall-derived TF) and several studies in the past have documented its role in the plaque thrombogenicity. At the end of the last century it was reported that, under physiological conditions, TF positive microparticles (MPs) circulate in the blood (blood-borne TF) and their number further increases in pathological conditions. These microparticles, which arise mainly from activated endothelial cells and monocytes, could fuse with other cells conferring them the ability to activate coagulation. Giesen et al. in 2000 showed the presence of TF in human platelets through this mechanism. Since then several papers have documented the presence of TF in human platelets by using different approaches. Despite this in-depth characterization, the platelet-associated TF is matter of controversy by some authors that, failing to identify TF in platelets, argue that the published data are artifacts, thus generating an ongoing debate. To date two main \u201ccellular entities\u201d may be responsible for the presence of TF in platelets: TF-positive MPs derived from different activated cell types, as proposed by Nemerson\u2019s group, and megakaryocytes that transferring TF mRNA to platelets make them autonomous in the synthesis of the protein. In this regard, ten years ago our group provided the evidence that human megakaryocytes contain TF mRNA. The platelet transcriptome derives from megakaryocytes through finely tuned mechanisms. The direct evidence that megakaryocytes transfer TF mRNA to platelets, however, is still missing; similarly, it has never been investigated whether megakaryocytes express TF protein that can be transferred to platelets; finally, the contribution of platelet TF to clot formation has never been assessed. To test these hypothesis we took advantage of a well-characterized human megakaryoblastic cell line, Meg-01, able to differentiate into megakaryocytes and to release platelets in vitro. This approach allowed us to analyze TF mRNA and protein expression during the differentiation from megakaryoblasts to megakaryocytes and in the released platelets in the complete absence of any other \u201ccontaminating\u201d cell that might be a source of microparticles. The expression of TF protein by Meg-01 and Meg-derived platelet (Meg-platelets) was confirmed in human megakaryocytes differentiated from CD34+ cells. Finally, TF mRNA was silenced in Meg-01 megakaryoblasts showing that megakaryocytes, Meg-platelets and MPs were almost devoid of TF. Using a combination of cell biology, flow cytometry, confocal microscopy and biochemical analyses here we show evidence for the first time that functionally active TF is expressed in human megakaryocytes, and that they transfer this protein to platelets and to microparticles (MPs). TF downregulation in megakaryoblasts by lentiviral shRNA particles almost completely abolished its expression in megakaryocytes, platelets and MPs. Furthermore, TF silencing leads to a decrease of the thrombin generation by megakaryocytes which is reflected on a decrease in thrombin generation by platelets, suggesting that the platelet-associated TF may contribute to the haemostatic capacity of whole blood. Taken together all these data shoe the evidence that human platelets and MPs carry a megakaryocyte-derived TF which is functionally active and able to trigger thrombin generation. Finally, this in vitro model may be used to investigate the modification in platelet transcriptome and functionality observed in several diseases, such as cardiovascular disease, diabetes and cancer

Prevalence of low antithrombin levels in preeclamptic women and perinatal outcome

Objective. The aim of this study is to evaluate the prevalence of low antithrombin levels in our population in order to assess an intervention trial feasibility. Methods. This is a retrospective study. A database was created by using queries to find out medical records of patients requiring hospitalization for preeclampsia or gestational hypertension or superimposed preeclampsia to chronic hypertension at Modena University Hospital between June 2015 and July 2019. Results. We screened 11845 deliveries. Overall, 221 (1.9%) cases of preeclampsia were identified. Antithrombin level was available for 201 women, thus included in the analysis. Median antithrombin value was 87% (IQ range: 77-98). The prevalence of low antithrombin levels was 9%. Antithrombin < 80% was found in 21% of the subjects. The remnant showed normal values. Median antithrombin was significantly lower in severe respect with mild preeclampsia (83% ± 14 vs 89% ± 14, p = 0.003). The rate of small for gestational age was significantly higher in low antithrombin levels group (44.4% vs 22.4%, p = 0.042). Considering mean values, antithrombin levels were also significantly lower in case of small for gestational age (84% ± 14 vs 89% ± 14; p = 0.040). Conclusions. In our population, low antithrombin levels (1 in 10 patients) were associated with severity of preeclampsia, namely with small for gestational age babies. Data suggest this subpopulation as a better target for trials assessing the efficacy of antithrombin supplementation

Immune checkpoint inhibitors in oncogene-addicted non-small cell lung cancer: A systematic review and meta-analysis

Background: Treatment of oncogene-addicted non-small cell lung cancer (NSCLC) has been changed by the advent of tyrosine kinase inhibitors (TKIs). Albeit great benefits are achieved with target therapies, resistance invariably occurs and recourse to alternative treatments is unavoidable. Immune checkpoint inhibitors (ICIs) role and the best setting of immunotherapy administration in oncogene-driven NSCLC are matter of debate. Methods: We performed a systematic literature review through PubMed, in order to gather all the available information regarding ICI activity and efficacy in oncogene-addicted NSCLC, from both prospective trials and retrospective series. A meta-analysis of objective response rate in different molecular subgroups was provided. Combinatorial strategies including ICIs and related toxicities were also recorded. Results: Eighty-seven studies were included in the qualitative analysis. EGFR mutation may be a biomarker of poor response to single-agent ICIs (7% of EGFR-mutant NSCLC patients achieved disease response in prospective trials), while encouraging results have been shown with combination strategies. KRASmutated disease (response rate, RR, 22%) has different clinical and pathological characteristics, and the coexistence of additional mutations (e.g., STK11 or TP53) influence tumor microenvironment and response to immunotherapy. Other molecular alterations have been marginally considered prospectively, and data from clinical practice are variegated, given poor effectiveness of ICIs in ALK-rearranged disease (RR 9.5%, pooling the data of retrospective studies) or some encouraging results in BRAF-(RR 25%, retrospective data) or MET-driven one (with estimations conditioned by the presence of both exon 14 skipping mutations and gene amplification in reported series). Conclusions: In oncogene-addicted NSCLC (with the exception of KRAS-mutated), ICIs are usually administered at the failure of other treatment options, but administering single-agent immunotherapy in later disease phases may limit its efficacy. With the progressive administration of TKIs and ICIs in early-stage disease, molecular characterization will become fundamental in this setting

Immune checkpoint inhibitors in oncogene-addicted non-small cell lung cancer: A systematic review and meta-analysis

BACKGROUND: Treatment of oncogene-addicted non-small cell lung cancer (NSCLC) has been changed by the advent of tyrosine kinase inhibitors (TKIs). Albeit great benefits are achieved with target therapies, resistance invariably occurs and recourse to alternative treatments is unavoidable. Immune checkpoint inhibitors (ICIs) role and the best setting of immunotherapy administration in oncogene-driven NSCLC are matter of debate. METHODS: We performed a systematic literature review through PubMed, in order to gather all the available information regarding ICI activity and efficacy in oncogene-addicted NSCLC, from both prospective trials and retrospective series. A meta-analysis of objective response rate in different molecular subgroups was provided. Combinatorial strategies including ICIs and related toxicities were also recorded. RESULTS: Eighty-seven studies were included in the qualitative analysis. EGFR mutation may be a biomarker of poor response to single-agent ICIs (7% of EGFR-mutant NSCLC patients achieved disease response in prospective trials), while encouraging results have been shown with combination strategies. KRAS-mutated disease (response rate, RR, 22%) has different clinical and pathological characteristics, and the co-existence of additional mutations (e.g., STK11 or TP53) influence tumor microenvironment and response to immunotherapy. Other molecular alterations have been marginally considered prospectively, and data from clinical practice are variegated, given poor effectiveness of ICIs in ALK-rearranged disease (RR 9.5%, pooling the data of retrospective studies) or some encouraging results in BRAF-(RR 25%, retrospective data) or MET-driven one (with estimations conditioned by the presence of both exon 14 skipping mutations and gene amplification in reported series). CONCLUSIONS: In oncogene-addicted NSCLC (with the exception of KRAS-mutated), ICIs are usually administered at the failure of other treatment options, but administering single-agent immunotherapy in later disease phases may limit its efficacy. With the progressive administration of TKIs and ICIs in early-stage disease, molecular characterization will become fundamental in this setting

Potential biomarkers for neuroinflammation and neurodegeneration at short and long term after neonatal hypoxic-ischemic insult in rat.

Background: Hypoxic-ischemic (HI) encephalopathy causes life-long morbidity and premature mortality in term neonates. Therapies in addition to whole-body cooling are under development to treat the neonate at risk for HI encephalopathy, but are not a quickly measured serum inflammatory or neuronal biomarkers to rapidly and accurately identify brain injury in order to follow the efficacy of therapies. Methods: In order to identify potential biomarkers for early inflammatory and neurodegenerative events after neonatal hypoxia-ischemia, both male and female Wistar rat pups at postnatal day 7 (P7) were used and had their right carotid artery permanently doubly occluded and exposed to 8% oxygen for 90 min. Sensory and cognitive parameters were assessed by open field, rotarod, CatWalk, and Morris water maze (MWM) test. Plasma and CSF biomarkers were investigated on the acute (24 h and 72 h) and chronic phase (4 weeks). Brains were assessed for gene expression analysis by quantitative RT-PCR Array. Results: We found a delay of neurological reflex maturation in HI rats. We observed anxiolytic-like baseline behavior in males more than females following HI injury. HI rats held on the rotarod for a shorter time comparing to sham. HI injury impaired spatial learning ability on MWM test. The CatWalk assessment demonstrated a long-term deficit in gait parameters related to the hind paw. Proinflammatory biomarkers such as IL-6 in plasma and CCL2 and TNF-\u3b1 in CSF showed an upregulation at 24 h after HI while other cytokines, such as IL-17A and CCL5, were upregulated after 72 h in CSF. At 24 h post-injury, we observed an increase of Edn1, Hif1-\u3b1, and Mmp9 mRNA levels in the ipsilateral vs the contralateral hemisphere of HI rats. An upregulation of genes involved with clotting and hematopoietic processes was observed 72 h post-injury. Conclusions: Our work showed that, in the immature brain, the HI injury induced an early increased production of several proinflammatory mediators detectable in plasma and CSF, followed by tissue damage in the hypoxic hemisphere and short-term as well as long-lasting neurobehavioral deficits

Postmenopausal hormone therapy in BRCA gene mutation carriers: to whom and which?

Introduction: Risk-reducing-salpingo-oophorectomy (RRSO) inevitably leads BRCA mutation carriers to premature menopause. Areas covered: To evaluate the existing evidence for use of postmenopausal hormone therapy (HT) in BRCAmc, after RRSO or menopause occurring naturally, for both breast cancer (BC) survivors and those without BC. Expert opinion: All BC survivors are excluded from any HT treatment: in other BRCAmc, before 51 years of age the benefits of HT overcome the risks after RRSO and/or premature ovarian insufficiency (POF). After 51 years of age, it is important to treat only women with important vasomotor symptoms, after the failure of alternative therapies. Estrogens-only therapy plays a key role in hysterectomized women (HW). In the case of an intact uterus (UW), associations with the lowest dose of progestins/natural progesterone derivatives have to be preferred, as progestins has been shown to play an important role in BC transformation, especially in BRCA1mc. No studies have been performed in BRCAmc with regard to ‘progestin-free’ HT, in particular the old tibolone (both in HW and UW) and the new tissue-selective estrogen complex (in UW). However, preliminary data obtained from the general population are reassuring about the use of these ‘progestin-free’ preparations and BC safety

Vaginal alpha-lipoic acid shows an anti-inflammatory effect on the cervix, preventing its shortening after primary tocolysis. A pilot, randomized, placebo-controlled study

Introduction: Inflammation might be an important underlying cause of preterm birth. Our aim is to explore whether vaginal administration α-lipoic acid reduces cervical inflammation and shortening after primary tocolysis. Materials and methods: Singleton pregnancies between 24–30 weeks remaining undelivered after hospitalization for preterm labor were randomly allocated to placebo (20 women, 15 analyzed) or vaginal ALA 400 mg (active ingredient 10 mg) daily (20 women, 17 analyzed) for 30 days. A cervical swab to quantify pro-inflammatory (IL1, IL2, IL6, IL8, TNFα) and anti-inflammatory (IL4, IL10) cytokines as well as transvaginal ultrasound cervical length measurement (CL) were performed before and after treatment. Results: The % changes of pro-inflammatory cytokines do not differ between treatment groups, while IL4 significantly increases by vaginal ALA in comparison to placebo (118.0 ± 364.3% versus 29.9 ± 103.5%, p = 0.012). Combined anti-inflammatory cytokines show same trend (292.5 ± 208.5% versus 64.5 ± 107.4, p = 0.03). CL remains similar in vaginal ALA group (from 23.1 ± 6.6 to 20.80 ± 7.9 mm), while it significantly decreased in placebo group (from 20.4 ± 6.5 to 13.8 ± 7.5 mm, p < 0.001 versus Baseline; p = 0.003 versus vaginal ALA). Conclusion: Vaginal ALA significantly stimulates anti-inflammatory ILs in the cervix of undelivered women after a preterm labor episode. This effect is associated with a stabilization of the CL

Astrocyte function is affected by aging and not Alzheimer's disease: A preliminary investigation in hippocampi of 3xTg-AD mice

Old age is a risk factor for Alzheimer's disease (AD), which is characterized by hippocampal impairment together with substantial changes in glial cell functions. Are these alterations due to the disease progression or are they a consequence of aging? To start addressing this issue, we studied the expression of specific astrocytic and microglial structural and functional proteins in a validated transgenic model of AD (3×Tg-AD). These mice develop both amyloid plaques and neurofibrillary tangles, and initial signs of the AD-like pathology have been documented as early as three months of age. We compared male 3×Tg-AD mice at 6 and 12 months of age with their wild-type age-matched counterparts. We also investigated neurons by examining the expression of both the microtubule-associated protein 2 (MAP2), a neuronal structural protein, and the brain-derived neurotrophic factor (BDNF). The latter is indeed a crucial indicator for synaptic plasticity and neurogenesis/ neurodegeneration. Our results show that astrocytes are more susceptible to aging than microglia, regardless of mouse genotype. Moreover, we discovered significant agedependent alterations in the expression of proteins responsible for astrocyte-astrocyte and astrocyte-neuron communication, as well as a significant age-dependent decline in BDNF expression. Our data promote further research on the unexplored role of astroglia in both physiological and pathological aging

Aqueye optical observations of the Crab Nebula pulsar

We observed the Crab pulsar in October 2008 at the Copernico Telescope in Asiago - Cima Ekar with the optical photon counter Aqueye (the Asiago Quantum Eye) which has the best temporal resolution and accuracy ever achieved in the optical domain (hundreds of picoseconds). Our goal was to perform a detailed analysis of the optical period and phase drift of the main peak of the Crab pulsar and compare it with the Jodrell Bank ephemerides. We determined the position of the main peak using the steepest zero of the cross-correlation function between the pulsar signal and an accurate optical template. The pulsar rotational period and period derivative have been measured with great accuracy using observations covering only a 2 day time interval. The error on the period is 1.7 ps, limited only by the statistical uncertainty. Both the rotational frequency and its first derivative are in agreement with those from the Jodrell Bank radio ephemerides archive. We also found evidence of the optical peak leading the radio one by ~230 microseconds. The distribution of phase-residuals of the whole dataset is slightly wider than that of a synthetic signal generated as a sequence of pulses distributed in time with the probability proportional to the pulse shape, such as the average count rate and background level are those of the Crab pulsar observed with Aqueye. The counting statistics and quality of the data allowed us to determine the pulsar period and period derivative with great accuracy in 2 days only. The time of arrival of the optical peak of the Crab pulsar leads the radio one in agreement with what recently reported in the literature. The distribution of the phase residuals can be approximated with a Gaussian and is consistent with being completely caused by photon noise (for the best data sets).Comment: 7 pages, 7 figures. Accepted for publication in Astronomy and Astrophysic