The mechanism of cell-damaging reactive oxygen generation by colloidal fullerenes

Because of the ability to induce cell death in certain conditions, the fullerenes (C₆₀) are potential anticancer and toxic agents. The colloidal suspension of crystalline C₆₀ (nano-C₆₀, nC₆₀) is extremely toxic, but the mechanisms of its cytotoxicity are not completely understood. By combining experimental analysis and mathematical modelling, we investigate the requirements for the reactive oxygen species (ROS)-mediated cytotoxicity of different nC₆₀ suspensions, prepared by solvent exchange method in tetrahydrofuran (THF/nC₆₀) and ethanol (EtOH/nC₆₀), or by extended mixing in water (aqu/nC₆₀). With regard to their capacity to generate ROS and cause mitochondrial depolarization followed by necrotic cell death, the nC₆₀ suspensions are ranked in the following order: THF/nC₆₀>EtOH/nC₆₀>aqu/nC₆₀. Mathematical modelling of singlet oxygen (¹O₂) generation indicates that the ¹O₂-quenching power (THF/nC₆₀nC₆₀nC₆₀) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause cell damage. These data could have important implications for toxicology and biomedical application of colloidal fullerenes.     

Beneficial effects of donor‐specific transfusion on renal allograft outcome

Jovi?i? S, Le?ai? V, Simonovi? R, Djukanovi? L. Beneficial effects of donor‐specific transfusion on renal allograft outcome.
Clin Transplant 2011: 25: 317–324. © 2010 John Wiley & Sons A/S. Abstract: Introduction: Donor‐specific transfusion (DST) is claimed to improve graft survival in living kidney transplantation. The aim of this study was to determine the influence of DST on the incidence of acute rejection (AR), graft function and survival in the early and late post‐transplantation period in transfusion‐naïve patients. Methods: Three patient groups were compared: group 1 received DST (n = 18), group 2 patients received no transfusion prior to surgery (n = 13) and group 3 consisted of 132 randomly transfused patients. The DST protocol consisted of infusion of fresh whole donor blood (3 × 150 mL) at two‐wk intervals accompanied by three d of azathioprine. All patients were grafted within one month after the third DST. Triple drug immunosuppression based on cyclosporine A was given to all patients. Results: DST and polytransfused patients experienced significantly less AR compared with group 2 patients. Two‐yr graft function was significantly better in patients in groups 1 and 3 compared with group 2. Although similar eight‐yr patient and graft survival was found in all the groups, delayed graft function patients had the longest graft half‐life. Conclusion: DST imposes a significant beneficial effect on the incidence of AR, DGF and graft function during the first post‐transplantation year in transfusion‐naïve patients receiving standard immunosuppression therapy.     

Novel duplication on chromosome 16 (q12.1-q21) associated with behavioral disorder, mild cognitive impairment, speech delay, and dysmorphic features: case report

We report on the 10-year follow-up and clinical, cytogenetic, and molecular investigation of a girl admitted for evaluation because of speech delay, learning difficulties, aggressive behavior, and dysmorphic facial features that included high forehead, round face, epicanthic folds, low-set dysplastic ears, flat nasal bridge, long flat philtrum, thin upper lip, small mouth, and short neck. The analysis of high-resolution GTG- and CTG-banding chromosomes suggested a de novo direct duplication of 16q12-q21 region and fluorescence in situ hybridization analysis with whole-chromosome specific 16 probe confirmed that the duplicated genetic material originated from the chromosome 16. Subsequently, array-based comparative genomic hybridization analysis with a≈75 kb resolution showed a 9.92 Mb gain on the long arm of chromosome 16 at bands q12.1 through q21. To the best of our knowledge, this is the first case of duplication 16q12.1q21 described in literature. Several genes within the duplicated region are possibly correlated with clinical features present in our patient. Clinical and cytogenetic findings were compared with the small number of reported patients with pure duplications 16q, partially overlapping the one in our patient. Clinical phenotype seems to be distinctive between the proximal-intermediate and intermediate-distal regions of the long arm of the chromosome 16. In particular, we observed a set of dysmorphic features that could present a characteristic dup 16q11.2-q13 phenotype. The present study illustrates the advantages of an integrative approach using both conventional and molecular techniques for the precise characterization and genotype-phenotype correlation in patients with dysmorphism, behavioral problems, and learning difficulties.     

Age-dependent morphometrical changes in the thymus of male propranolol-treated rats.

In order to elucidate a putative role of neurally derived noradrenaline in the thymus development, and in maintenance of adult thymus structure, sexually immature male rats (21-day-old at the beginning of treatment) and young adult animals (75-day-old on the beginning of treatment) were treated with the non-selective beta-adrenoceptor antagonist propranolol (0.40 mg/100 g BW/day, s.c.) for 15 consecutive days, and their thymuses were analyzed stereologically. The effects of beta-adrenoceptor blockade were much more pronounced in sexually immature than in adult rats. In immature propranolol-treated rats the thymus size and volumes of both the main compartments (cortex and medulla) were significantly decreased reflecting, at least partly, a reduction in the overall number of thymocytes. Furthermore, in both the cortical subcompartments (outer and deep cortex) the mean diameter of thymocytes was increased. However, in adult rats exposed to propranolol treatment, only the volume of interlobular connective tissue was enlarged, whereas in the outer part of the cortex the mean thymocyte diameter was increased. These results indicate that the lack of sympathetic input (via beta-adrenoceptors) during the prepubertal period of development diminishes the normal thymus growth and/or accelerates the thymic involution that starts at puberty, immediately after its maximum size is reached, while it is less significant for the maintenance of the thymus size and structure in adults. Additionally, they suggest that distinct cell types, as well as thymocyte subsets, are sensitive to lack of beta-adrenoceptor-mediated influences in sexually immature and adult rats.     

Immunomodulatory actions of central ghrelin in diet-induced energy imbalance

We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalance-induced inflammation. Rats were subjected for four weeks to three different dietary regimes: normal (standard food), high-fat (standard food with 30% lard) or food-restricted (70%, 50%, 40% and 40% of the expected food intake in 1st, 2nd, 3rd and 4th week, respectively). Compared to normal-weight controls, starved, but not obese rats had significantly higher levels of proinflammatory cytokines (TNF, IL-1β, IFN-γ) in the blood. When compared to normally fed animals, the hearts of starved and obese animals expressed higher levels of mRNAs encoding proinflammatory mediators (TNF, IL-1β, IL-6, IFN-γ, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-β remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 μg/day) for five consecutive days significantly reduced TNF, IL-1β and IFN-γ levels in the blood of starved rats, as well as TNF, IL-17 and IL-12p40 mRNA expression in the hearts of obese rats. Conversely, ICV ghrelin increased the levels of IFN-γ, IL-17, IL-12p35 and IL-12p40 mRNA in the heart tissue of food-restricted animals. This was associated with an increase of immunosuppressive ACTH/corticosterone production in starved animals and a decrease of the immunostimulatory adipokine leptin both in food-restricted and high-fat groups. Ghrelin activated the energy sensor AMP-activated protein kinase (AMPK) in the hypothalamus and inhibited extracellular signal-regulated kinase (ERK) in the hearts of obese, but not starved rats. Therefore, central ghrelin may play a complex role in energy imbalance-induced inflammation by modulating HPA axis, leptin and AMPK/ERK signaling pathways.     

A novel biochemically salvageable animal model of hyperammonemia devoid of N-acetylglutamate synthase

All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-acetylglutamate synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-l-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. Founder NAGS knockout heterozygous mice were obtained from the trans-NIH Knock-Out Mouse Project. Genotyping of the mice was performed by PCR and confirmed by Western blotting of liver and intestine. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags(-/-)) pups and the rescued animals were characterized. We observed an 85% survival rate of Nags(-/-) mice when they were given intraperitoneal injections with NCG and Cit during the newborn period until weaning and supplemented subsequently with both compounds in their drinking water. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 h. In addition to hyperammonemia, interruption of rescue supplementation was associated with elevated plasma glutamine, glutamate, and lysine, and reduced citrulline, arginine, ornithine and proline levels. We conclude that NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.     

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8⁺ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), first emerged in late 2019 in China (1). SARS-CoV-2 exhibits extremely efficient human-to-human transmission, the new pathogen rapidly spread worldwide, and within months it caused a global pandemic, changing daily life for billions of people. The COVID-19 case fatality rate of ∼2–5% makes the development of countermeasures a global priority. In fact, the development of COVID-19 vaccine candidates is advancing at an international level with unprecedented speed. About 1 y after the first known cases of COVID-19, we can account for >80 SARS-CoV-2–specific vaccines in clinical evaluations and >10 candidate vaccines already in phase III trials (2–4). However, we still lack information on the key immune mechanisms needed for protection against COVID-19. A better understanding of the types of immune response elicited upon natural SARS-CoV-2 infections has become an essential component to assess the promise of various vaccination strategies (5).The SARS-CoV-2 spike (S) protein serves as the most important target antigen for vaccine development based on preclinical research on candidate vaccines against SARS-CoV or Middle East respiratory syndrome coronavirus (MERS-CoV). The trimeric S protein is a prominent structure at the virion surface and essential for SARS-CoV-2 cell entry. As a class I viral fusion protein, it mediates virus interaction with the cellular receptor angiotensin-converting enzyme 2 (ACE2), and fusion with the host cell membrane, both key steps in infection. Thus, infection can be prevented by S-specific antibodies neutralizing the virus (6–9).Among the front-runner vaccines are new technologies such as messenger RNA (mRNA)-based vaccines and nonreplicating adenovirus vector vaccines (10–13). First reports from these SARS-CoV-2-S–specific vaccines in phase 1/2 clinical studies demonstrated acceptable safety and promising immunogenicity profiles, and by now data from large phase 3 clinical trials show promising levels of protective efficacy (4, 12–14). In December 2020, the first mRNA-based COVID-19 vaccines received emergency use authorization or conditional licensing by the US Food and Drug Administration and European Medicines Agency (11, 15, 16). By March 2021, two adenovirus vector-based COVID-10 vaccines had been approved by regulatory authorities (17, 18). This is good news because efficacious vaccines will provide a strategy to change SARS-CoV-2 transmission dynamics. In addition, multiple vaccine types will be advantageous to meet specific demands across different target populations. This includes the possibility of using heterologous immunization strategies depending on an individual’s health status, boosting capacities, and the need for balanced humoral and Th1-directed cellular immune responses.MVA, a highly attenuated strain of vaccinia virus originating from growth selection on chicken embryo tissue cultures, shows a characteristic replication defect in mammalian cells but allows unimpaired production of heterologous proteins (19). At present, MVA serves as an advanced vaccine technology platform for developing new vector vaccines against infectious disease including emerging viruses and cancer (20). In response to the ongoing pandemic, the MVA vector vaccine platform allows rapid generation of experimental SARS-CoV-2–specific vaccines (21). Previous work from our laboratory addressed the development of an MVA candidate vaccine against MERS with immunizations in animal models demonstrating the safety, immunogenicity, and protective efficacy of MVA-induced MERS-CoV S-antigen–specific immunity (22–25). Clinical safety and immunogenicity of the MVA-MERS-S candidate vaccine was established in a first-in-human phase I clinical study under funding from the German Center for Infection Research (DZIF) (26).Here, we show that a recombinant MVA produces the full-length S protein of SARS-CoV-2 as ∼190- to 200-kDa N-glycosylated protein. Our studies confirmed cleavage of the mature full-length S protein into an amino-terminal domain (S1) and a ∼80- to 100-kDa carboxyl-terminal domain (S2) that is anchored to the membrane. When tested as a vaccine in BALB/c mice, recombinant MVA expressing the S protein induced SARS-CoV-2–specific T cells and antibodies, and robustly protected vaccinated animals against lung infection upon SARS-CoV-2 challenge.