The Impact of Risk-Adjusted Heparin Regimens on the Outcome of Patients with COVID-19 Infection. A Prospective Cohort Study.

BACKGROUND: According to recent guidelines, all hospitalized patients with COVID-19 should receive pharmacological prophylaxis for venous thromboembolism (VTE), unless there are specific contraindications. However, the optimal preventive strategy in terms of intensity of anticoagulation for these patients is not well established. OBJECTIVES: To investigate the impact of individualized regimens of enoxaparin on the development of VTE and on the risk of major bleeding complications during hospitalization in patients with COVID-19 infection. METHODS: All consecutive patients admitted to the medical wards of six Italian hospitals between 15 September and 15 October 2020 with COVID-19 infection of moderate severity were administered enoxaparin in subcutaneous daily doses adjusted to the Padua Prediction Score stratification model: No heparin in patients scoring less than 4, 4000 IU daily in those scoring 4, 6000 IU in those scoring 5, and 8000 in those scoring six or more. Objective tests were performed in patients developing clinical symptoms of deep vein thrombosis and/or pulmonary embolism. Bleeding complications were defined according to the ISTH classification. RESULTS: From the 154 eligible patients, enoxaparin was administered in all: 4000 IU in 73 patients, 6000 IU in 53, and 8000 IU in the remaining 28. During the course of hospitalization, 27 patients (17.5%) died. VTE developed in 14 of the 154 patients (9.1%; 95% CI, 4.6% to 13.6%), and was fatal in 1. Major bleeding complications developed in 35 patients (22.7%; 95% CI, 16.1% to 29.3%), and were fatal in 8. CONCLUSIONS: Despite the use of risk-adjusted doses of enoxaparin, the rate of VTE events was consistent with that reported in contemporary studies where fixed-dose low-molecular-weight heparin was used. The unexpectedly high risk of bleeding complications should induce caution in administering enoxaparin in doses higher than the conventional low ones.

Antithrombotic and Anti-Inflammatory Effects of Fondaparinux and Enoxaparin in Hospitalized COVID-19 Patients: The FONDENOXAVID Study.

BACKGROUND: Since the outbreak of novel coronavirus SARS-CoV2 around the world, great attention has been paid to the effects of such antithrombotic drugs as heparinoids, because they have antiviral action in vitro and antithrombotic actions in vivo. We conducted a retrospective analysis in inpatients with confirmed COVID-19 on the anti-inflammatory and antithrombotic effects of enoxaparin and fondaparinux at prophylactic doses. METHODS: This retrospective cohort study used patients with confirmed COVID-19 during the first months of the Italian outbreak from February 18 to April 30, 2020. Our aim was to compare clinical characteristics, prophylactic treatment, markers of inflammation, and thrombotic outcomes in inpatients positive for SARS-CoV2 during hospitalization associated with thromboprophylaxis with enoxaparin (40 mg or 60 mg once daily) or fondaparinux (2.5 mg once daily). Statistical analysis was conducted with using MatLab R2016B and ad hoc functions. RESULTS: There were no significatant differences in clinical characteristics between patients that used enoxaparin or fondaparinux as thromboprophylaxis for SARS-CoV2. No differences were found in D-dimer and fibrinogen levels either, which were used as markers of inflammation during the infection at testing on admission and after 3 weeks.Significant differences in CRP, IL6, and LDH were found in patients after 21 days' treatment. DISCUSSION: Increased levels of fibrinogen and D-dimer in patients with confirmed COVID-19 have been reported in several studies. Our results showed that anti-inflammatory effects of fondaparinux and enoxaparin after 3 weeks of prophylactic treatment were similar when levels of fibrinogen and D-dimer were considered. Furthermore, levels of CRP showed a decrease in patients treated with enoxaparin and fondaparinux, although the decrease in the fondaparinux group seems to be more relevant.

Thromboprofilaxys With Fondaparinux vs. Enoxaparin in Hospitalized COVID-19 Patients: A Multicenter Italian Observational Study.

Importance: The use of anticoagulant therapy with heparins decreased mortality in hospitalized patients with severe coronavirus disease 2019 (COVID-19). Even if enoxaparin and fondaparinux have the same clinical indication for venous thromboembolism (VTE) prevention; to date, there are no data about the use of fondaparinux in terms of safety, effectiveness, and impact on clinical prognosis among COVID-19 patients. Objective: To evaluate the safety, effectiveness, and clinical impact of VTE prophylaxis with fondaparinux and enoxaparin among COVID-19 patients hospitalized in internal medicine units. Design, Setting, and Participants: This was a retrospective multicenter observation study, including consecutive symptomatic patients with laboratory-proven COVID-19 admitted to internal medicine units of five Italian hospitals from 15th February to 15th March 2020. Main Outcomes and Measures: The primary safety outcome was the composite of major bleeding and clinically relevant non-major bleeding; the primary effectiveness outcome was the composite of all events classified as pulmonary embolism and deep venous thrombosis. The secondary effectiveness outcome included acute respiratory distress syndrome and all-cause death. Results: Among 120 COVID-19 patients enrolled in the study, 74 were taking enoxaparin (4,000 or 6,000 units/day) and 46 fondaparinux (2.5 units/day). No statistically significant difference in demographic and laboratory and clinical characteristics between the two groups has been shown. During a median follow-up of 32 (interquartile range: 14-51) days, the cumulative incidence rates of VTE and bleeding events on pharmacological thromboprophylaxis with heparins were 19% and 8%, respectively. The incidence of both VTE (6.5 vs. 13.5%; P = 0.36) and bleeding events (6.5 vs. 4.1%; P = 0.68) did not show a significant difference between COVID-19 patients on fondaparinux compared with those on enoxaparin therapy. The regression model for the risk of outcome events according to different VTE prophylaxis drugs did not show significant differences. Conclusions and Relevance: Although these results need confirmation by prospective studies including a larger population, our study provides preliminary evidence of a safe and efficacy use of fondaparinux for VTE prophylaxis in hospitalized COVID-19 patients.

Fondaparinux Use in Patients With COVID-19: A Preliminary Multicenter Real-World Experience.

The use of heparin has been shown to decrease the mortality in hospitalized patients with severe COVID-19. The aim of our study was to evaluate the clinical impact of venous thromboembolism prophylaxis with fondaparinux versus enoxaparin among 100 hospitalized COVID-19 patients. The incidence of pulmonary embolism, deep venous thrombosis, major bleeding (MB), clinically relevant non-MB, acute respiratory distress syndrome, and in-hospital mortality was compared between patients on fondaparinux versus enoxaparin therapy. The 2 groups were homogeneous for demographic, laboratory, and clinical characteristics. In a median follow-up of 28 (IQR: 12-45) days, no statistically significant difference in venous thromboembolism (14.5% vs. 5.3%; P = 0.20), MB and clinically relevant non-MB (3.2% vs. 5.3%, P = 0.76), ARDS (17.7% vs. 15.8%; P = 0.83), and in-hospital mortality (9.7% vs. 10.5%; P = 0.97) has been shown between the enoxaparin group versus the fondaparinux group. Our preliminary results support the hypothesis of a safe and effective use of fondaparinux among patients with COVID-19 hospitalized in internal medicine units.

Individualized 3D-printed templates for high-dose-rate interstitial multicathether brachytherapy in patients with breast cancer.

PURPOSE: High-dose-rate, multicatheter interstitial brachytherapy is technically complex and operator-dependent, requiring lengthy training and specialized skills. Furthermore, until the advent of contouring on computerized tomography (CT) images, difficulties existed in locating the target volume precisely. The present article reports the results of a study that aimed at producing and validating a 3D-printed template to aid in target volume localization for multicatheter interstitial brachytherapy in patients with breast cancer. METHODS AND MATERIALS: Thirteen patients, candidates for accelerated partial breast irradiation or boost, were enrolled in the study. The target volume was defined on CT slices, and a template with empty spaces corresponding to the target volume projection on the patient's skin was produced by a 3D printer. The procedure was compared with the standard method followed in our center (1) visually, by assessing overlap between the target volume projections on the patient's skin, (2) by X-ray findings, and (3) by intraclass correlation coefficient. RESULTS: Visual assessment and X-ray findings showed the 3D-printed target volume always fell within the standard volume in all 13 patients. The intraclass correlation coefficient indicated moderate agreement for both the medial and the lateral skin projections. CONCLUSIONS: The 3-D printed templates constitute a quick, easy, and reliable method to localize the target volume for high-dose-rate interstitial multicathether brachytherapy in patients with breast cancer and can safely be used in clinical practice.

TSH receptor and thyroid-specific gene expression in human skin.

Experimental evidence suggests that in autoimmune thyroid diseases (AITDs) the skin is a target of autoantibodies against thyroid-specific antigens; however, the role of these autoantibodies in skin alterations remains unclear. To gain insight into the function of nominally thyroid-specific genes in skin, we analyzed the expression of thyroid-stimulating hormone-receptor (TSH-R), thyroglobulin (Tg), sodium iodide symporter (NIS), and thyroperoxidase (TPO) genes in normal human skin biopsies and cultured primary keratinocytes and dermal fibroblasts. The results revealed the presence of all the transcripts in skin biopsies. However, in keratinocytes and fibroblasts, only TSH-R messenger RNA was always detected. Western blot and immunohistochemical analyses of skin specimens confirmed the presence of TSH-R protein in keratinocytes and fibroblasts. Moreover, TSH treatment induced the proliferation of cultured keratinocytes and fibroblasts and increased keratinocyte intracellular cAMP. Finally, affinity-purified IgGs from serum of patients affected by Graves' disease, but not by chronic lymphocytic thyroiditis, stimulated cAMP accumulation in cultured keratinocytes, as well as their proliferation. In conclusion, the expression of thyroid-specific genes in cultured keratinocytes and fibroblasts and the mitogenic effects of TSH and IgGs on these cells support the concept that autoantibodies against thyroid-specific antigens may contribute to cutaneous symptoms in AITDs.

Computed tomography for excision cavity localization and 3D-treatment planning in partial breast irradiation with high-dose-rate interstitial brachytherapy.

BACKGROUND AND PURPOSE: When high-dose-rate brachytherapy is used for partial breast irradiation (PBI) precise pre-implant definition of planning target volume (PTV) and implant geometry is required. After implantation, accurate PTV localization, catheter reconstruction and optimization of dose distribution are needed for good PTV coverage and dose conformity. We applied image-guidance using computed tomography (CT) for pre-implant PTV definition and post-implant dosimetry. MATERIALS AND METHODS: In 54 patients implant geometry was designed by external beam virtual simulation. A template was placed over dummy beam digitally reconstructed radiographs displaying PTV. Needle entrance and exit points were defined and marked on the patient's skin to serve as landmarks during implantation. After implantation, in 46/54 patients PTV was defined, catheters were reconstructed and active lengths in the catheters were specified using CT-based-3D planning system. Dosimetry was performed with a Plato-Nucletron treatment planning system. RESULTS: Post-implantation CT visualized precise catheter placement with respect to the PTV in all patients. CT-based treatment planning provided good coverage of PTV and homogeneous dose distribution. CONCLUSIONS: In post-operative PBI with high-dose-rate brachytherapy CT-based pre-implant definition of implant geometry ensures adequate PTV coverage. After implantation, CT-based 3D-treatment planning software ensures exact PTV localization and catheter reconstruction, and dose distribution optimization.

Acute pressor and hormonal effects of beta-endorphin at high doses in healthy and hypertensive subjects: role of opioid receptor agonism.

CONTEXT: The opioid system is involved in blood pressure regulation in both normal humans and patients with essential hypertension. OBJECTIVE: The objective of the study was to investigate the effects of a high-dose infusion of beta-endorphin, an opioid peptide, on blood pressure and on the hormonal profile in healthy subjects and in hypertensive patients and the mediation played by opioid receptor agonism. DESIGN, SETTING, AND PARTICIPANTS: According to a randomized double-blind design, 11 healthy subjects (controls) and 12 hypertensive inpatients (mean age, 38.9 and 40.4 yr, respectively) received 1-h iv infusion of beta-endorphin (250 mug/h) and, on another occasion, the same infusion protocol preceded by the opioid antagonist naloxone (8 mg). MAIN OUTCOME MEASURES: Hemodynamic and hormonal measurements were performed at established times during the infusion protocols. RESULTS: At baseline, circulating beta-endorphin, norepinephrine, and endothelin-1 in hypertensive patients were significantly (P < 0.05) higher than in controls. In controls, beta-endorphin reduced blood pressure (P < 0.01) and circulating norepinephrine (P < 0.02) and increased plasma atrial natriuretic factor (P < 0.003) and GH (P < 0.0001). In hypertensive patients, beta-endorphin decreased systemic vascular resistance (P < 0.0001), blood pressure (P < 0.0001), and plasma norepinephrine (P < 0.0001) and endothelin-1 (P < 0.0001) and raised circulating atrial natriuretic factor (P < 0.0001), GH (P < 0.0001), and IGF-I (P < 0.0001). These hemodynamic and hormonal responses to beta-endorphin in hypertensive patients were significantly (P < 0.0001) greater than in controls but were annulled in all individuals when naloxone preceded beta-endorphin infusion. CONCLUSIONS: High doses of beta-endorphin induce hypotensive and beneficial hormonal effects in humans, which are enhanced in essential hypertension and are mediated by opioid receptors.

The bromodomain-containing protein Bdf1p acts as a phenotypic and transcriptional multicopy suppressor of YAF9 deletion in yeast.

It was observed previously that the deletion of the open reading frame YNL107w (YAF9) was highly pleiotropic in yeast and caused defective growth phenotypes in the presence of several unrelated inhibitors, including caesium chloride. We have selected multicopy extragenic suppressor genes, revealing that this phenotype can be suppressed by overdosing the transcription factors BDF1 and GAT1 in the yaf9Delta strain. We focused our analysis on suppression by BDF1 and performed a genome-wide transcript analysis on a yaf9Delta strain, compared with the wild-type and BDF1-suppressed strains. YAF9 deletion has a clear effect on transcription and leads to modulation of the level of expression of several genes. Transcription of a considerable portion of the underexpressed genes is restored to wild-type levels in the BDF1-suppressed strain. We show by chromatin immunoprecipitation that both Yaf9p and Bdf1p bind to promoters of some of these genes and that the level of H3 and H4 acetylation at one of these promoters is significantly lowered in the yaf9 deleted strain, compared with the wild-type and the BDF1-suppressed strains.

Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4.

Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.

Engineering a V(2) vasopressin receptor agonist- and regulator of G-protein-signaling-sensitive G protein.

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to G(s)alpha. Furthermore, G(s)alpha is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V(2) receptor with a series of chimeric G protein alpha subunits in which the C-terminal 6-12 amino acids of G(i1)alpha were replaced with the equivalent sequence of G(s)alpha allowed robust vasopressin-stimulated [(35)S]GTPgammaS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V(2) receptor and either G(s)alpha or G(i1)alpha. However, it produced a concentration-dependent stimulation of V(max) for a V(2) receptor-G(i1)alpha/Gs6alpha fusion protein. This construct bound [(3)H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V(2) receptor. RGS1 enhanced vasopressin stimulation of V(2) receptor-G(i1)alpha/G(s)6alpha in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both V(max) of the GTPase activity and the observed K(m) for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V(2) receptor agonist ligands and may be amenable to many other G(s)alpha-coupled receptors.