Characterization of metabolic alterations in the lean metabolically unhealthy alpha defensin transgenic mice.

Inflammation is consistently linked to dysmetabolism. In transgenic mice (Def+/+) model the neutrophilic peptide, alpha defensin, proved atherogenic. This phenotype occurred despite favorable cholesterol and glucose levels, and lower body weight and blood pressure. In this study, integration of metabolic&behavioral phenotyping system, endocrine, biochemical and mitochondrial assessment, pathological and immunohistochemical tests, and multiple challenge tests was established to explore the metabolic impact of alpha defensin. Compared to the control group, Def+/+ mice exhibited lower total energy expenditure and carbohydrate utilization, and higher fat oxidation. Their ACTH-cortisol and thyroid profiles were intact. Intriguingly, they had low levels of glucagon, with high ammonia, uric acid, triglyceride, and lactate. Mitochondrial evaluations were normal. Overall, defensin-induced hypoglucagonemia is associated with lipolysis, restricted glucose oxidation, and enhanced wasting. Def+/+ mice may be a useful model for studying the category of lean, apparently metabolically healthy, and atherosclerotic phenotype, with insight into a potential inflammatory-metabolic link.

Alleviation of Hepatic Steatosis by Alpha-Defensin Is Associated with Enhanced Lipolysis.

Background and Objectives: The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods: A cohort of transgenic C57BL/6JDef+/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef+/+) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results: The Def+/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def+/+ mice. Conclusions: Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.

Human Neutrophil α-Defensins 1-3 Are Upregulated in the Microenvironment of Fibrotic Liver.

Background and Objectives: Neutrophil infiltration is an established signature of Non-Alcoholic Fatty Liver Disease (NAFLD) and Steatohepatitis (NASH). The most abundant neutrophilic peptide, alpha-defensin, is considered a new evolving risk factor in the inflammatory milieu, intimately involved in lipid mobilization. Our objective is to assess for potential association between alpha-defensin immunostains and NAFLD severity. Materials and Methods: We retrospectively investigated the liver biopsies of NAFLD/NASH patients, obtained at Hillel Yaffe Medical center between the years 2012 and 2016. Patients' characteristics were recorded, including relevant blood tests at the time of biopsy. Each biopsy was semi-quantitatively scored using NAFLD Activity Score (NAS) and NASH fibrosis stage. The biopsies were immunostained for alpha-defensin. The precipitation of alpha-defensin was correlated to NAS and fibrosis. Results: A total of 80 biopsies were evaluated: male ratio 53.2%, mean age 44.9 ± 13.2 years, 54 had fibrosis grades 0-2, and 26 were grade 3-4. Conventional metabolic risk factors were more frequent in the high-grade fibrosis group. Immunostaining for alpha-defensin disclosed higher intensity (a.u.) in grade 3-4 fibrosis relative to grades 0-2, 25% vs. 6.5%, p < 0.05, respectively. Moreover, alpha-defensin staining was nicely co-localized with fibrosis. Conclusions: In our group of NASH/NAFLD patients, higher metabolic risk profile was associated with higher fibrosis grade. Immunostaining for alpha-defensin showed patchy intense staining concordant with high fibrosis, nicely co-localized with histological fibrosis. Whether alpha-defensin is a profibrotic risk factor or merely risk marker for fibrosis must be clarified in future studies.

SARS-CoV-2 Infection-Blocking Immunity Post Natural Infection: The Role of Vitamin D.

OBJECTIVE AND AIM: The extent of the protection against SARS-CoV-2 conferred by natural infection is unclear. Vitamin D may have a role in the interplay between SARS-CoV-2 infection and the evolving acquired immunity against it. We tested the correlation between baseline 25(OH) D content and both the reinfection rate and the anti-spike protein antibody titer following COVID-19 infection. Methods A retrospective observational survey that included a large convalescent COVID-19 population of subjects insured by the Leumit HMO was recorded between 1 February 2020 and 30 January 2022. Inclusion criteria required at least one available 25(OH)D level prior to enlistment. The association between 25(OH)D levels, the rate of breakthrough infection, and the anti-spike protein antibody titer was evaluated. Results A total of 10,132 COVID-19 convalescent subjects were included, of whom 322 (3.3%) sustained reinfection within a one-year follow-up. In the first 8 months after recovery, the reinfected patients were characterized by a higher incidence of low 25(OH)D levels (<30 ng/mL, 92% vs. 84.8%, p < 0.05), while during the following three months, the incidence of low 25(OH)D levels was non-significantly higher among PCR-negative convalescent subjects compared to those reinfected (86% vs. 81.7, p = 0.15). By multivariate analysis, age > 44 years (OR-0.39, 95% CI: 0.173-0.87, p = 0.02) and anti-spike protein antibody titer > 50 AU/mL (0.49, 95% CI: 0.25-0.96, p = 0.04) were inversely related to reinfection. No consistent correlation with vitamin D levels was observed among the 3351 available anti-spike protein antibody titers of convalescent subjects. However, the median anti-spike protein antibody titers tended to increase over time in the vitamin D-deficient group. Conclusion Higher pre-infection 25(OH)D level correlated with protective COVID-19 immunity during the first 8 months following COVID-19 infection, which could not be explained by anti-spike protein antibody titers. This effect dissipated beyond this period, demonstrating a biphasic 25(OH)D association that warrants future studies.

Correlation between Baseline 25(OH) Vitamin D Levels and Both Humoral Immunity and Breakthrough Infection Post-COVID-19 Vaccination.

Objective: Vaccines against COVID-19 induce specific antibodies whose titer is perceived as a reliable correlate of protection. Vitamin D confers complex regulatory effects on the innate and adaptive immunity. In this study, we explored a plausible impact of baseline vitamin D content on achieved immunity following COVID-19 vaccination. Methods: A retrospective observational study comprising 73,254 naïve subjects insured by the Leumit Health Service HMO, who were vaccinated between 1 February 2020 and 30 January 2022, with one available vitamin D level prior to vaccination, was performed. The association between 25(OH) vitamin D levels, SARS-CoV-2 antibody titer, and post-vaccination PCR results were evaluated. Results: Of the study population, 5026 (6.9%) tested positive for COVID-19. The proportion of low 25(OH)D levels (<30 ng/mL) was significantly higher in the PCR-positive group (81.5% vs. 79%, p < 0.001). Multivariate analysis showed a higher incidence of breakthrough infection among non-smokers [1.37 (95% CI 1.22−1.54, p < 0.001)] and lower incidences among subjects with sufficient 25(OH)D levels (>30 ng/mL) [0.87 (95% CI 0.79−0.95, p­0.004)], hyperlipidemia [0.84 (95% CI 0.76−0.93, p < 0.001], depression [OR-0.87 (95% CI: 0.79−0.96, p < 0.005], socio-economic status >10 [0.67 (95% CI 0.61−0.73, p < 0.001)], and age >44 years. SARS-CoV-2 antibody titers were available in 3659 vaccinated individuals. The prevalence of antibody titers (<50 AU) among PCR-positive subjects was 42% compared to 28% among PCR-negative subjects (p < 0.001). Baseline 25(OH)D levels showed an inverse relation to total antibody titers. However, no association was found with an antibody titer <50 AU/mL fraction. Conclusion Baseline 25(OH)D levels correlated with the vaccination-associated protective COVID-19 immunity. Antibody titers <50 AU/mL were significantly linked to breakthrough infection but did not correlate with 25(OH)D levels.

Association between tissue human neutrophil peptide 1-3 levels and cardiovascular phenotype: a prospective, longitudinal cohort study.

OBJECTIVE: Inflammation is associated with atherogenesis. Although a higher neutrophil count is associated with the plaque burden, the role of neutrophil activation is unclear. Human neutrophil peptides 1-3 (HNP1-3) are a risk factor for atherogenesis in bench models and are elevated in human atheromas. This study aimed to examine the association between skin HNP1-3 deposition and the severity of coronary artery disease (CAD), including long-term outcomes. METHODS: HNP1-3 levels were immunohistochemically quantified in skin biopsies, which were prospectively taken from 599 consecutive patients before clinically indicated coronary angiography. Established cardiovascular risk factors and blood markers for atheroinflammation were obtained. CAD severity and the incidence of repeat revascularization and mortality at 48 months of follow-up were assessed in relation to HNP1-3 levels. RESULTS: The risk of CAD was independently associated with age and HNP1-3 in the entire cohort (F = 0.71 and F = 7.4, respectively). Additionally, HNP1-3 levels were significantly associated with myocardial necrosis (R = 0.26). At the follow-up, high HNP1-3 levels negatively affected mortality (19.54%) and recurrent revascularization (8.05%). CONCLUSION: HNP1-3 tissue deposition is positively associated with the severity of CAD, myonecrosis, and long-term sequelae. HNP1-3 levels may be suppressed using colchicine.

Morphine Use in ST-Elevation Myocardial Infarction With Downstream P2Y12 Receptor Blockers-Insight From Observational Study.

BACKGROUND AND AIMS: Morphine use for patients presenting with NSTE-ACS is associated with excess mortality. However, the role of morphine in STE-ACS is ill characterized. We have recently confirmed direct prothrombotic effect of morphine using murine models. We sought to explore whether morphine use in STE-ACS patients, used to be scheduled for downstream P2Y12 blockers, is negatively associated with procedural and clinical outcomes. METHODS: A single-center, observational retrospective analysis enrolling 130 non-randomized stable patients sustaining STE-ACS as their first manifestation of coronary disease, who presented between December 2010 and June 2013. All were managed by early invasive approach. Of study patients, 55 were treated by morphine, and 75 were not. All were administered downstream P2Y12 blockers according to an already abandoned local policy. Outcomes evaluated included TIMI grade flow, thrombus burden, ST-segment resolution, myocardial function by echocardiography, and cardiovascular death. RESULTS: Morphine administration was associated with a significantly higher incidence of impaired final TIMI grade flow (TIMI < 3, 40% vs 4%, P < .05), lower incidence of ST-segment resolution >70% (40.7% vs 76.5%, P < .05), and a higher incidence of moderate or severe systolic dysfunction (48.1% vs 29.1%, P < .05) compared with morphine naive patients. Interestingly, the overall mortality rate was higher in the morphine-treated group (18% vs 5.3%, P < .05). CONCLUSIONS AND RELEVANCE: Morphine administration combined with the downstream P2Y12 blockers practice signify a group with a higher occurrence of impaired myocardial reperfusion and cardiovascular death despite established on-time primary angioplasty.

Divergent impacts of tocilizumab and colchicine in COVID-19-associated coagulopathy: the role of alpha-defensins.

Patients who are severely affected by coronavirus disease 2019 (COVID-19) may develop a delayed onset 'cytokine storm', which includes an increase in interleukin-6 (IL-6). This may be followed by a pro-thrombotic state and increased D-dimers. It was anticipated that tocilizumab (TCZ), an anti-IL-6 receptor monoclonal antibody, would mitigate inflammation and coagulation in patients with COVID-19. However, clinical trials with TCZ have recorded an increase in D-dimer levels. In contrast to TCZ, colchicine reduced D-dimer levels in patients with COVID-19. To understand how the two anti-inflammatory agents have diverse effects on D-dimer levels, we present data from two clinical trials that we performed. In the first trial, TCZ was administered (8 mg/kg) to patients who had a positive polymerase chain reaction test for COVID-19. In the second trial, colchicine was given (0·5 mg twice a day). We found that TCZ significantly increased IL-6, α-Defensin (α-Def), a pro-thrombotic peptide, and D-dimers. In contrast, treatment with colchicine reduced α-Def and Di-dimer levels. In vitro studies show that IL-6 stimulated the release of α-Def from human neutrophils but in contrast to colchicine, TCZ did not inhibit the stimulatory effect of IL-6; raising the possibility that the increase in IL-6 in patients with COVID-19 treated with TCZ triggers the release of α-Def, which promotes pro-thrombotic events reflected in an increase in D-dimer levels.

Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis.

Atherosclerosis, the predominant cause of death in well-resourced countries, may develop in the presence of plasma lipid levels within the normal range. Inflammation may contribute to lesion development in these individuals, but the underlying mechanisms are not well understood. Transgenic mice expressing α-def-1 released from activated neutrophils develop larger lipid and macrophage-rich lesions in the proximal aortae notwithstanding hypocholesterolemia caused by accelerated clearance of α-def-1/low-density lipoprotein (LDL) complexes from the plasma. The phenotype does not develop when the release of α-def-1 is prevented with colchicine. However, ApoE-/- mice crossed with α-def-1 mice or given exogenous α-def-1 develop smaller aortic lesions associated with reduced plasma cholesterol, suggesting a protective effect of accelerated LDL clearance. Experiments were performed to address this seeming paradox and to determine if α-def-1 might provide a means to lower cholesterol and thereby attenuate atherogenesis. We confirmed that exposing ApoE-/- mice to α-def-1 lowers total plasma cholesterol and decreases lesion size. However, lesion size was larger than in mice with total plasma cholesterol lowered to the same extent by inhibiting its adsorption or by ingesting a low-fat diet. Furthermore, α-def-1 levels correlated independently with lesion size in ApoE-/- mice. These studies show that α-def-1 has competing effects on atherogenesis. Although α-def-1 accelerates LDL clearance from plasma, it also stimulates deposition and retention of LDL in the vasculature, which may contribute to development of atherosclerosis in individuals with normal or even low plasma levels of cholesterol. Inhibiting α-def-1 may attenuate the impact of chronic inflammation on atherosclerotic vascular disease.

Acute diagonal-induced ST-elevation myocardial infarction and electrocardiogram-guidance in the era of primary coronary intervention: New insights into an old tool.

BACKGROUND: Previous studies, published before the advent of primary reperfusion, described the electrocardiographic features of ST-segment elevation myocardial infarction (STEMI) caused by total diagonal artery occlusion, as demonstrated at pre-discharge coronary angiography. We aimed to assess the electrocardiographic and echocardiographic features in STEMI unequivocally attributed to a diagonal lesion in the era of primary coronary intervention. METHODS: The electrocardiograms and echocardiograms of patients sustaining STEMI caused by diagonal artery involvement were compared with those of patients with STEMI attributed to proximal or mid left anterior descending artery (LAD) lesions. ST-segment deviations were measured at four different points in each lead and analyzed against TIMI flow and SNuH score. The electrocardiographic and echocardiographic features of each group were mapped. RESULTS: In contrast to previous studies claiming an ever-present incidence of at least 1-mm ST-segment elevation in leads I and aVL with diagonal STEMI, we report 86% of any ST-elevation in leads I, aVL and V2 (64-71% for ST-elevation >1 mm). Both higher SNuH score and pre-intervention TIMI flow were associated with larger lateral ST-elevations (85.7% and 86.4-95.5%, respectively). Higher prevalence of ST-depression in the inferior leads reflecting reciprocal changes was observed in patients with diagonal-induced STEMI (57-76% vs. 24-51% in LAD obstructions, p <0.05). CONCLUSION: The most sensitive and predictive sign for acute ischemia was any degree of ST-deviation measured 1 mm beyond the J point. ST-elevations in I, aVL and V2, sparing V3-V5, strongly favor isolated diagonal lesion. Proximal LAD lesion lacking ST-segment elevations in leads I and aVL is primarily due to wraparound LAD anatomy.

Neutrophil α-defensins promote thrombosis in vivo by altering fibrin formation, structure, and stability.

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.

α-Defensins Induce a Post-translational Modification of Low Density Lipoprotein (LDL) That Promotes Atherosclerosis at Normal Levels of Plasma Cholesterol.

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def(+/+)). Accelerated Def(+/+) mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def(+/+) to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def(+/+) mice prevented these outcomes. The same outcome was obtained by treating Def(+/+) mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis.

Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice.

Persistent intracerebral hemorrhage (ICH) is a major cause of death and disability after traumatic brain injury (TBI) for which no medical treatment is available. Delayed bleeding is often ascribed to consumptive coagulopathy initiated by exposed brain tissue factor. We examined an alternative hypothesis, namely, that marked release of tissue-type plasminogen activator (tPA) followed by delayed synthesis and release of urokinase plasminogen activator (uPA) from injured brain leads to posttraumatic bleeding by causing premature clot lysis. Using a murine model of severe TBI, we found that ICH is reduced in tPA(-/-) and uPA(-/-) mice but increased in PAI-1(-/-) mice compared with wild-type (WT) mice. tPA(-/-), but not uPA(-/-), mice developed a systemic coagulopathy post-TBI. Tranexamic acid inhibited ICH expansion in uPA(-/-)mice but not in tPA(-/-) mice. Catalytically inactive tPA-S(481)A inhibited plasminogen activation by tPA and uPA, attenuated ICH, lowered plasma d-dimers, lessened thrombocytopenia, and improved neurologic outcome in WT, tPA(-/-), and uPA(-/-) mice. ICH expansion was also inhibited by tPA-S(481)A in WT mice anticoagulated with warfarin. These data demonstrate that protracted endogenous fibrinolysis induced by TBI is primarily responsible for persistent ICH and post-TBI coagulopathy in this model and offer a novel approach to interrupt bleeding.