Antibody Response to the BNT162b2 mRNA COVID-19 Vaccine in Subjects with Prior SARS-CoV-2 Infection

Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.     

Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE^−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Atherosclerotic plaque rupture is the leading cause of myocardial infarction and stroke (1, 2). Studies assessing the correlation between calcium scores and cardiovascular events have demonstrated a predictive power that is superior to and independent from that of lipid scores (3, 4). Additionally, clinical imaging studies have revealed that the risk of plaque rupture is further heightened by the presence of small, “spotty” calcifications, or microcalcifications (5, 6), and cardiovascular risk is inversely correlated with the size of calcific deposits, quantified as a calcium density score (7). Indeed, computational modeling has demonstrated that, while large calcifications can reinforce the fibrous cap (8), microcalcifications (typically 5 to 15 μm in diameter) uniquely mediate an increase in mechanical stress of the relatively soft, collagen-rich fibrous cap (9–12).Histologic studies have revealed the presence of cell-derived vesicles within calcifying atherosclerotic lesions (13–16). The inflammatory environment of the atherosclerotic lesion can induce vascular smooth muscle cells (vSMCs) to take on an osteochondrogenic phenotype and release calcifying extracellular vesicles (EVs) (17–19). Macrophages have also been shown to release procalcifying vesicles (20, 21). Thus, just as bone formation is hypothesized to be an active, cell-driven process (22, 23), mediated by calcifying matrix vesicles, atheroma-associated calcification may similarly be initiated by the production and aggregation of calcifying EVs (11, 20, 24–28).One proposed strategy for halting pathologic calcification has been the use of bisphosphonates (BiPs). BiPs are analogs of pyrophosphate (29), a naturally occurring compound derived in vivo from adenosine triphosphate (ATP) (30). Pyrophosphate binds to calcium phosphate and inhibits calcification via physicochemical mechanisms, namely, by blocking calcium and phosphate ions from forming crystals, preventing crystal aggregation, and preventing mineral transformation from amorphous calcium phosphate to hydroxyapatite (29). BiPs were identified as pyrophosphate analogs that, unlike pyrophosphate itself, resist enzymatic hydrolysis. A second, distinct property of BiPs is the ability to inhibit bone resorption via biological activity directed against osteoclasts following osteoclast endocytosis of the BiP molecule adsorbed to the surface of bone (29, 31). First-generation, or nonnitrogen-containing BiPs, are incorporated into nonhydrolyzable ATP analogs, and induce osteoclast apoptosis by limiting ATP-dependent enzymes. In contrast, nitrogen-containing BiPs inhibit farnesyl pyrophosphate synthetase and thereby induce osteoclast apoptosis (31).In vivo animal investigations have been performed to explore the potential for BiPs to inhibit cardiovascular calcification. Studies of first-generation BiPs revealed that the doses required to inhibit cardiovascular calcification also critically compromised normal bone mineralization (29, 32). However, newer, nitrogen-containing BiPs effectively arrested cardiovascular calcification in animal models at doses that did not compromise bone formation (32). Further, while it has been proposed that BiP treatment modifies cardiovascular calcification via its impact on bone-regulated circulating calcium and phosphate levels, a study in uremic rats demonstrated that BiP treatment inhibited medial aortic calcification with no significant change in plasma calcium and phosphate levels (33). The same study demonstrated that BiP treatment inhibited calcification of explanted rat aortas, indicating that BiPs can act directly on vascular tissue, independent of bone metabolism (33).Retrospective clinical data examining the effect of BiP therapy on cardiovascular calcification has demonstrated conflicting findings and intriguing paradoxes. In women with chronic kidney disease, BiP therapy decreased the mortality rate for patients without a prior history of cardiovascular disease (34), but for those patients with a history of prior cardiovascular events, BiP therapy was associated with an increased mortality rate (35). In another study, BiP therapy correlated with a lower rate of cardiovascular calcification in older patients (>65 y), but a greater rate in younger patients (<65 y) (36). These clinical findings motivated our study, in which we sought to further understand how BiP therapy impacts cardiovascular outcomes. Given that cardiovascular calcification, and especially the presence of microcalcification, is a strong and independent risk factor for adverse cardiac events, and BiPs are prescribed to modulate pathologies of mineralization, we hypothesize that BiPs modulate cardiovascular outcomes by altering the dynamics of cardiovascular calcification.EVs are smaller than the resolution limits of traditional microscopy techniques, hindering studies into the mechanisms of calcification nucleation and growth. We previously developed an in vitro collagen hydrogel platform that allowed the visualization of calcific mineral development mediated by EVs isolated from vSMCs (24). Using superresolution microscopy, confocal, and electron microscopy techniques, we showed that calcification requires the accumulation of EVs that aggregate and merge to build mineral. Collagen serves as a scaffold that promotes associations between EVs that spread into interfibrillar spaces. The resultant mineral that forms within the collagen hydrogel appears spectroscopically similar to microcalcifications in human tissues and allows the study of these structures on the time scale of 1 wk. In this study, we utilized this three-dimensional (3D) acellular platform to examine the direct effect of ibandronate, a nitrogen-containing BiP, on the EV-directed nucleation and growth of microcalcifications, a process that cannot be isolated from cellular and tissue-level mechanisms in a more complex, in vivo system. In parallel, we utilized a mouse model of atherosclerosis to assess the effect of ibandronate therapy on plaque-associated calcification, comparing mineral morphologies between the in vitro and in vivo samples. We hypothesize that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Understanding the EV-specific action of BiPs is imperative both to develop anticalcific therapeutics targeting EV mineralization and to understand one potential mechanism driving the cardiovascular impact of BiPs used in clinical settings.     

Prevalence of endotoxemia after surgery and its association with ICU length of stay

Introduction

The aim of this observational study was to investigate the prevalence of endotoxemia after surgery and its association with ICU length of stay.

Methods

102 patients admitted to a university ICU after surgery were recruited. Within four hours of admission, functional data were collected and APACHE II severity score calculated. Arterial blood samples were taken and endotoxemia was measured by chemiluminescence (Endotoxin Activity (EA)). Patients were stratified according to their endotoxin levels (low, intermediate and high) and according to their surgical procedures. Differences between endotoxin levels were assessed by ANOVA, accepting P < 0.05 as significant. Data are expressed as mean ± SD.

Results

EA levels were low in 68 (66%) patients, intermediate in 17 (17%) and high in 17 (17%). Age (61 ± 17 years) and APACHE II score 8.3 ± 3.7 (P = 0.542) were not significantly different in the three EA groups. Functional parameters on admission were similar between EA groups: white blood cells 11093 ± 4605 cells/mm³ (P = 0.385), heart rate 76 ± 16 bpm (P = 0.898), mean arterial pressure 88.8 ± 13.6 mmHg (P = 0.576), lactate 1.18 ± 0.77 mmol/L (P = 0.370), PaO₂/FiO₂ 383 ± 109 mmHg (P = 0.474). Patients with high levels of EA were characterized by longer length of stay in the ICU: 1.9 ± 3.0 days in the low EA group, 1.8 ± 1.4 days in intermediate and 5.2 ± 7.8 days in high group (P = 0.038).

Conclusions

17% of our patients were characterized by high levels of endotoxemia as assessed by EA assay, despite their low level of complexity on admission. High levels of endotoxin were associated with a longer ICU length of stay.     

Divergent roles of growth factors in the GnRH regulation of puberty in mice

Pubertal onset, initiated by pulsatile gonadotropin-releasing hormone (GnRH), only occurs in a favorable, anabolic hormonal milieu. Anabolic factors that may signal nutritional status to the hypothalamus include the growth factors insulin and IGF-1. It is unclear which hypothalamic neuronal subpopulation these factors affect to ultimately regulate GnRH neuron function in puberty and reproduction. We examined the direct role of the GnRH neuron in growth factor regulation of reproduction using the Cre/lox system. Mice with the IR or IGF-1R deleted specifically in GnRH neurons were generated. Male and female mice with the IR deleted in GnRH neurons displayed normal pubertal timing and fertility, but male and female mice with the IGF-1R deleted in GnRH neurons experienced delayed pubertal development with normal fertility. With IGF-1 administration, puberty was advanced in control females, but not in females with the IGF-1R deleted in GnRH neurons, in control males, or in knockout males. These mice exhibited developmental differences in GnRH neuronal morphology but normal number and distribution of neurons. These studies define the role of IGF-1R signaling in the coordination of somatic development with reproductive maturation and provide insight into the mechanisms regulating pubertal timing in anabolic states.     

Appropriateness of angiography for suspected coronary artery disease

The aim of this study was to assess the appropriate use of diagnostic catheterizations (DC) for the patients with suspected coronary artery disease performed in Iran. The Electronic Health Record System database and manual review of files were utilised to collect data between 2012 and 2014. Patients were categorized in three groups as appropriate, uncertain, and inappropriate usage of DC and the logistic regression was used to investigate the relationships between variables. One-quarter of the 2458 angiographies were rated as inappropriate, out of which 99% had no previous stress test. The rate of inappropriate DC between various hospitals were approximately the same. The regression showed that some risk factors (Sex, high cholesterol, smoking, chronic heart failure, renal failure, diabetes) were significantly associated with inappropriate rate.     

Human colonic myogenic dysfunction induced by mucosal lipopolysaccharide translocation and oxidative stress

Background

Impairment of gastrointestinal motility is frequently observed in patients with severe infection.

Aim

To assess whether exposure of human colonic mucosa to pathogenic lipopolysaccharide affects smooth muscle contractility.

Methods

Human colonic mucosa and submucosa were sealed between two chambers, with the luminal side facing upwards and covered with Krebs solution, with or without lipopolysaccharide from a pathogenic strain of Escherichia coli (O111:B4; 1000 ng/mL), and with the submucosal side facing downwards into Krebs. The solution on the submucosal side was collected following 30-min mucosal exposure to Krebs without (N-undernatant) or with lipopolysaccharide (lipopolysaccharide undernatant). Undernatants were tested for lipopolysaccharide and hydrogen peroxide levels and for their effects on smooth muscle cells in the presence of catalase, indomethacin or MG132.

Results

Smooth muscle cells incubated with N-undernatant had a maximal contraction of 32 ± 5% that was reduced by 62.9 ± 12% when exposed to lipopolysaccharide undernatant. Inhibition of contraction was reversed by catalase, indomethacin and MG132. Lipopolysaccharide levels were higher in the lipopolysaccharide undernatant (2.7 ± 0.7 ng/mL) than in N-undernatant (0.45 ± 0.06 ng/mL) as well as hydrogen peroxide levels (133.75 ± 15.9 vs 82 ± 7.5 nM respectively).

Conclusions

Acute exposure of colonic mucosa to pathogenic lipopolysaccharide impairs muscle cell contractility owing to both lipopolysaccharide mucosal translocation and production of free radicals.