The Synergistic Effect of Time of Exposure, Distance and No Use of Personal Protective Equipment in the Determination of SARS-CoV-2 Infection: Results of a Contact Tracing Follow-Up Study in Healthcare Workers.

The aim of this study is to assess the effect of contact time, contact distance and the use of personal protective equipment on the determination of SARS-CoV-2 infection in healthcare workers (HCWs). This study consists of an analysis of data gathered for safety reasons at the Sapienza Teaching Hospital Policlinico Umberto I in Rome through the surveillance system that was put into place after the worsening of the COVID-19 pandemic. The studied subjects consist of HCWs who were put under health surveillance, i.e., all employees who were in contact with subjects who were confirmed to have tested positive for SARS-CoV-2. The HCWs under surveillance were monitored for a period encompassing ten days after the date of contact, during which they undertook nasopharyngeal swab tests analysed through RT-PCR (RealStar® SARS-CoV-2 Altona Diagnostic-Germany). Descriptive and univariate analyses have been undertaken, considering the following as risk factors: (a) no personal protective equipment use (PPE); (b) Distance < 1 m between the positive and contact persons; (c) contact time > 15'. Finally, a Cox regression and an analysis of the level of synergism between factors, as specified by Rothman, were carried out. We analysed data from 1273 HCWs. Of these HCWs, 799 (62.8%) were females, with a sample average age of 47.8 years. Thirty-nine (3.1%) tested positive during surveillance. The overall incidence rate was 0.4 per 100 person-days. Time elapsed from the last exposure and a positive RT-PCR result ranged from 2 to 17 days (mean = 7, median = 6 days). In the univariate analysis, a distance <1 m and a contact time > 15' proved to be risk factors for the SARS-CoV-2 infection, with a hazard ratio (HR) of 2.62 (95% CI: 1.11-6.19) and 3.59 (95% IC: 1.57-8.21), respectively. The synergism analysis found the highest synergism between the "no PPE use" x "Contact time". The synergy index S remains strongly positive also in the analysis of the factors "no PPE use" x "Distance" and "Time of contact" x "Distance". This study confirms the absolute need to implement safety protocols during the pandemic and to use the correct PPE within health facilities in order to prevent SARS-CoV-2 infection. The analysis shows that among the factors considered (contact time and distance, no use of PPE), there is a strong synergistic effect.

Digoxin and Platelet Activation in Patients With Atrial Fibrillation: In Vivo and In Vitro Study.

Background Digoxin use was shown to be associated with an increased risk of cardiovascular events in atrial fibrillation ( AF ). We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation. Methods and Results Post hoc analysis of a prospective study of anticoagulated patients with AF . Patients were divided into 2 groups balanced for age, sex, and cardiovascular risk factors: digoxin users (n=132) and nonusers (n=388). Urinary excretion of 11-dehydro-thromboxane B2 (TxB2), a marker of platelet activation, and serum digoxin concentration ( SDC ) were measured. In vitro experiments were performed on platelets from healthy subjects and AF patients, which were incubated with scalar doses of digoxin (0.6-2.4 ng/mL) with or without prestimulation with a sub-threshold of collagen. Median 11-dehydro-TxB2 was 105.0 ( interquartile range, 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 ( interquartile range, 0.40-1.00) ng/mL. Urinary 11-dehydro-TxB2 and SDC were correlated ( rs=0.350, P<0.001). Patients in the upper tertile of SDC showed higher 11-dehydro-TxB2 compared with non-digoxin users ( P=0.019). In vitro study showed an increased basal platelet activation in patients with AF compared with healthy subjects . Digoxin (2.4 ng/mL) induced calcium mobilization, PAC -1 (procaspase-activating compound 1) and platelet aggregation in AF patients but not in healthy subjects . After pretreatment with a sub-threshold of collagen, digoxin dose-dependent induced calcium mobilization, arachidonic acid release, TxB2 biosynthesis, PAC -1 and soluble platelet selectin expression, and platelet aggregation, which were inhibited by antibody against digoxin. Conclusions We found a significant in vivo correlation between SDC and platelet activation. Supratherapeutic SDC increased in vitro platelet aggregation via calcium-related phospholipase A2 phosphorylation. Our findings may have clinical implications for AF patients treated with digoxin.

Relationship of PCSK9 and Urinary Thromboxane Excretion to Cardiovascular Events in Patients With Atrial Fibrillation.

BACKGROUND: Soluble proprotein convertase subtilisin/kexin type 9 (PCSK9) has been shown to be predictive of cardiovascular events (CVEs) in patients who are at high cardiovascular risk. No data on the effect of PCSK9 levels in patients with atrial fibrillation (AF) are available. OBJECTIVES: This study investigated the association between PCSK9 and CVEs in AF as well as the relationship between PCSK9 and urinary 11-dehydro-thromboxane B2 (11-dh-TxB2), a marker of platelet activation. METHODS: We conducted a prospective, single-center cohort study, including 907 patients with AF treated with vitamin K antagonists (3,865 patient-years), to assess CVEs, including fatal and nonfatal myocardial infarction, ischemic stroke, and cardiovascular death. At admission, plasma PCSK9 and urinary 11-dh-TxB2 (n = 852) were measured. The population was divided into tertiles of PCSK9 for the analysis. RESULTS: The mean age of patients was 73.5 ± 8.2 years, and 43.0% were women. At follow-up, 179 CVEs (4.6%/year) occurred: 43 (15.3%), 49 (15.5%), and 87 (28.0%) in the first, second, and third tertiles of PCSK9, respectively (log-rank test p = 0.009). Patients with CVEs had higher median PCSK9 compared with those without (1,500 pg/ml [IQR: 1,000 to 2,300 pg/ml] vs. 1,200 pg/ml [IQR: 827 to 1,807 pg/ml], respectively; p < 0.001). Multivariable Cox regression analysis showed that the third versus the first tertile of PCSK9 (hazard ratio: 1.640; 95% confidence interval: 1.117 to 2.407; p = 0.012), female sex, age, diabetes, smoking, heart failure, previous cerebrovascular and cardiac events, digoxin use, and total cholesterol to high-density lipoprotein cholesterol ratio were associated with CVEs. In 682 patients not treated with antiplatelet therapy, circulating PCSK9 and 11-dh-TxB2 were significantly correlated (Spearman's rho: 0.665; p < 0.001). CONCLUSIONS: Plasma PCSK9 levels are associated with an increased risk of CVEs in patients with AF. The direct correlation between PCSK9 and 11-dh-TxB2 suggests PCSK9 as a mechanism potentially implicated in platelet activation.

Plasma levels of matrix metalloproteinases 2 and 9 correlate with histological grade in breast cancer patients.

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in the process of tumor invasion and metastasis that are found throughout tissues and also in the plasma. The aim of this study was to investigate whether the evaluation of plasma concentrations of MMPs 2, 3 and 9 may have clinical significance in breast cancer. Therefore, sera obtained from 80 patients with breast neoplasia (50 carcinomas and 30 fibroadenomas) were collected before and 96 h after surgery and the concentrations of MMPs 2, 3 and 9 were quantified using an enzyme-linked immunosorbent assay (ELISA). The mean expression level of MMP 2 was significantly higher in carcinoma compared with that in fibroadenoma patients, while there was no significant difference for MMPs 3 and 9. In addition, the group of carcinoma patients was analyzed in order to compare the mean values for each MMP obtained before and after surgery. However, the differences between pre- and post-surgery values for all three MMPs were not statistically significant. Furthermore, the plasma levels of each MMP were correlated with certain clinicopathological parameters of the tumors and we observed a significant and direct correlation between the concentrations of MMPs 2 and 9 and tumor histological grade. These data suggest that the quantification of plasma MMP 2 and MMP 9 levels may provide additional clinical information of the tumor and it is, therefore, a possible prognostic index for breast cancer.

Ischemia and apoptosis in an animal model of permanent infarct-related artery occlusion.

Apoptosis is a pathologic feature of cardiomyocytes in acute myocardial infarction (AMI) and heart failure. The temporal course of apoptosis in the peri-infarct area in the weeks following an AMI is still uncompletely defined. In order to study the time course of apoptosis after AMI, 16 rabbits underwent left coronary artery ligation and were sacrificed at 16, 26, 35, and 56 days after surgery. Increased apoptotic rate (AR) was observed at in the peri-infarct region than in remote myocardium (5.4% [2.5-9.6] vs 0.4% [0.1-0.9], respectively, P<0.001) and than in sham-operated cases (0.01% [0-0.02], P<0.001). A gradual decrease of AR in the peri-infarct region was observed over time with a 90% reduction at 8 weeks after coronary ligation.

Myocardial expression of survivin, an apoptosis inhibitor, in aging and heart failure. An experimental study in the spontaneously hypertensive rat.

BACKGROUND: Apoptosis plays a major role in the transition to heart failure (HF) in systemic hypertension although the underlying mechanisms are still unclear. The aim of this study was to determine the relationship between apoptosis, left ventricular remodeling, heart failure and the myocyte expression of survivin, an inhibitor of apoptosis. METHODS: Spontaneously hypertensive rats (SHR) were used as a model of hypertensive cardiopathy, and Wistar Kyoto Stars rats (WKY) were used as controls. Animals were allowed to survive up to 18 months of age. The animals underwent echocardiography (EDD, ESD and FS were measured). The median section of the heart was processed for in situ end-labeling of DNA fragmentation (TUNEL) and for survivin expression by immunohistochemistry. RESULTS: All SHR presented features of adverse cardiac remodeling. Apoptotic cells were increased in SHR compared with WKY, measured as apoptotic cells per high power field (1.08+/-0.43 vs. 0.27+/-0.15, P<0.001), and as apoptotic rate (0.16+/-0.06% vs. 0.04+/-0.02%, P<0.001). The incidence of apoptosis showed a positive correlation with unfavorable ventricular remodeling, assessed by echocardiogram. Survivin expression was found in all cases, but the survivin expression index was significantly lower in SHR vs. WKY (43+/-40% vs. 86+/-18%, respectively, P=0.014). Moreover the survivin expression index was inversely correlated with features of adverse remodeling (i.e., Heart Weight, R=-0.79, P<0.001) and with apoptosis (i.e., apoptotic rate, R=-0.52, P=0.050). CONCLUSION: Survivin myocardial expression in aging SHR is associated with reduced apoptosis and more favorable cardiac remodeling. Modulation of this pathway may prove beneficial in preventing pressure overload cardiac remodeling and heart failure.

Comparison of extracellular matrix and apoptotic markers between benign lesions and carcinomas in human breast.

The expression of the extracellular matrix-related genes, such as fibronectin, laminin and tenascin C, and apoptosis-related genes, such as bax, bcl2 and survivin, was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and by immunohistochemistry in normal breast tissue and benign and malignant breast tumors and then correlated to several clinical parameters: estrogen and progesterone receptors, Ki67, ErbB2, tumor size, lymph node status and grading. Seventy-three breast tissue samples were examined. After RNA extraction, an RT-PCR was performed to detect fibronectin, laminin, tenascin C, bax, bcl2 and survivin gene expression. Thirty-two samples were evaluated also by immunohistochemistry at the protein level to detect fibronectin, laminin, tenascin C, bax and survivin. We found a significant correlation (P=0.025) between fibronectin gene expression and lymph node status, and a significant negative correlation (P=0.049) between laminin gene expression and Ki67. In addition, we found a statistically significant increase in survivin transcription in malign tumors compared to fibroadenomas (P=0.024). The negative correlation between laminin transcription and Ki67 could suggest that laminin impacts negatively on tumor proliferation, and the positive correlation between fibronectin and lymph node status may lead to consider fibronectin as predictive of long distance metastasis.

Infarct-related artery occlusion, tissue markers of ischaemia, and increased apoptosis in the peri-infarct viable myocardium.

AIMS: Unfavourable cardiac remodelling often complicates acute myocardial infarction (AMI) as a result of increased cardiomyocyte apoptosis. It is currently unclear whether ongoing or recurrent ischaemia is an independent determinant for increased apoptosis in peri-infarct viable myocardium. METHODS AND RESULTS: In order to assess the link between infarct-related artery (IRA) occlusion, ischaemia, and apoptosis, 30 subjects dying 7-120 days after AMI (16 with IRA occlusion and 14 with patent IRA) and five control subjects were selected at autopsy. Cardiomyocytes were defined as apoptotic if co-expressing TUNEL and activated caspase-3. Expression of both hypoxia-inducible factor-1 and cyclo-oxygenase-2 was assessed in the peri-infarct myocardium and considered as tissue markers of ischaemia. Evidence of ischaemia was significantly more frequent in cases with IRA occlusion (53%) than in cases with patent IRA (15%) or control hearts (0%, P=0.026). The finding of IRA occlusion and markers of ischaemia identified cases with higher apoptotic rates (ARs) in the peri-infarct viable myocardium [12.2% (8.2-14.0), P<0.001 vs. others], whereas IRA occlusion without ischaemia was associated with lower AR, not significantly different from patent IRA [3.0% (1.0-7.9) vs. 2.2% (1.0-5.8), respectively, P=0.42] CONCLUSION: Ischaemia in the peri-infarct viable myocardium is present in over 50% of subjects dying late after AMI with IRA occlusion, and it is associated with increased apoptosis. Relief of ischaemia after AMI may prove of benefit in preventing apoptosis and its consequences.

Cyclooxygenase-2 overexpression is associated with a poor outcome in resected ampullary cancer patients.

PURPOSE: To identify potential prognostic molecular factors in ampullary adenocarcinoma that could be of significant importance. To this end, we examined the possible prognostic significance of cyclooxygenase-2 (Cox-2) and Survivin expression and the apoptotic index in a cohort of uniformly treated patients with ampullary cancer treated with radical surgical excision. EXPERIMENTAL DESIGN: The entry criteria were that the patients have a pathologic diagnosis of ampullary cancer which had been resected. Expression analysis for Cox-2 and Survivin was done by immunohistochemical staining. Apoptotic cells were identified by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. RESULTS: Thirty-nine tumor specimens from resected ampullary adenocarcinoma patients were included. By univariate analysis, overall survival was affected by Cox-2 expression and TUNEL staining (respectively, P = 0.0003 and 0.03). Survivin expression did not influence the overall survival in our patient population (P = 0.123). Patients' clinicopathologic features (gender, age, and T and N factors) did not influence outcome. In multivariate Cox regression analysis, Cox-2 expression (relative risk, 4.330; P = 0.005) was the only variable that significantly affected overall survival. CONCLUSIONS: The results of the present article provide, for the first time, evidence that Cox-2 expression, but not Survivin expression, may represent a significant prognostic factor after surgical resection in patients affected by cancer of the ampulla of Vater. Further studies are required to determine whether Cox-2 inhibitors may be useful for the therapy or prevention of ampullary carcinoma.

Hypoxia inducible factor-1 expression mediates myocardial response to ischemia late after acute myocardial infarction.

We report hypoxia-inducible factor-1 (HIF-1) expression in myocardium of patients with recent acute myocardial infarction (AMI), supporting the hypothesis of HIF-1 as a possible mediator of response to ischemia. A potential diagnostic role of determining tissue expression of HIF-1 as a marker of ischemia, and potential therapeutic implications by trying to modulate HIF-1 activity in order to promote beneficial effects of HIF-1 related genes (e.g. expression of vascular endothelial growth factor (VEGF)) may derive.

Increased apoptosis in remote non-infarcted myocardium in multivessel coronary disease.

BACKGROUND: Multivessel coronary disease after myocardial infarction is a major risk factor for unfavorable cardiac remodeling and death due to pump failure, but underlying pathophysiologic mechanisms are still uncompletely established. Post-infarction myocardial apoptosis has been recently implicated as a cause of ongoing cell loss leading to cardiac failure. Our aim was to assess the role of post-infarction myocardial apoptosis and pro-apoptotic factor expression in the non-infarcted remote myocardium of subjects with multivessel coronary disease. METHODS: Twenty-one males dying after recent myocardial infarction with permanent occlusion of the infarct-related artery were selected at autopsy. Apoptosis was assessed at viable myocardial regions remote from infarction by co-staining for in situ end-labeling of DNA fragmentation and cleaved caspase-3. Expression of pro-apoptotic factor bax and hypoxia-induced factor-1alpha was evaluated by immunohistochemistry. RESULTS: Subjects with multivessel disease (N=11) showed a significantly two-fold higher myocardial apoptosis in comparison to subjects with single vessel disease (N=10) (0.9% vs. 0.5%, p=0.013). Similarly, myocardial bax expression was increased in patients with multivessel disease (3.0% vs. 1.4%, p=0.029). Stratification for the number of diseased coronary vessels confirmed the association between extent of coronary disease and apoptotic rates (p=0.022). Even in subjects dying over 30 days after infarction multivessel disease remained predictive of enhanced myocardiocyte apoptosis at remote regions (p=0.033). CONCLUSIONS: Post-infarction myocardial apoptosis and bax expression in remote left ventricular regions are significantly increased in male patients with multivessel coronary disease in comparison to those with isolated infarct-related artery occlusion. These findings suggest that apoptotic cell loss in the viable non-infarcted myocardium, possibly due ongoing ischemia, may play a relevant role in the unfavorable clinical course typical of multivessel disease after myocardial infarction.

Survivin, bcl-2, bax, and bcl-X gene expression in sentinel lymph nodes from melanoma patients.

PURPOSE: The expression of apoptosis-related genes, such as survivin, bcl-2, bcl-X, and bax, has been evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry in sentinel lymph nodes (SLNs) from melanoma patients and then correlated to the outcome of patients. PATIENTS AND METHODS: Thirty-six SLNs were examined. After RNA extraction, an RT-PCR followed by Southern blot hybridization was performed to detect survivin, bcl-2, bcl-X, and bax mRNA. bcl-2, survivin, and bax gene expression was evaluated, whenever possible, also by immunohistochemistry at the protein level. RESULTS: We found a significant correlation (P <.005) between survivin expression and outcome of patients; in fact, 61.5% of patients expressing survivin gene progressed or died because of the disease, whereas 38.5% are currently disease-free. Among patients negative for survivin expression, 100% are disease-free after a median follow-up time of 52.9 months. We did not find a significant correlation between bcl-2, bax, and bcl-X gene expression and outcome of patients. In fact, these genes were found equally expressed in patients with disease progression and in disease-free patients. CONCLUSION: Our findings show a variable expression of apoptosis-related genes in SLNs of melanoma patients; more interestingly, we found that survivin expression correlates to outcome of patients in a statistically significant way, whereas the expression of other genes, such as bcl-2, bax, and bcl-X, did not seem to correlate to progression of disease. We suggest that the detection of survivin gene expression by RT-PCR in SLNs may be a useful prognostic indicator.

Retinoic acid inhibits fibronectin and laminin synthesis and cell migration of human pleural mesothelioma in vitro.

The effects of retinoic acid (RA) on cell replication, fibronectin and laminin synthesis, integrin expression and haptotactic migration of three mesothelioma cell cultures of different histotype, one epithelioid, one fibromatous and one biphasic, were evaluated. Cell growth was not affected by RA, while RA treatment decreased the synthesis of fibronectin and laminin and inhibited the migration of all three mesotheliomas on substrates of fibronectin and laminin; on the contrary, the expression of some integrins was not significantly modified by RA. These data indicate that RA may lead to a decrease of mesothelioma cell local invasion; this can correlate with a modification induced by RA on mesothelioma tumor progression in vivo.