Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy

Background SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. Methods Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. Results Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. Conclusions The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.Peer Reviewe

Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin plastic sections of infected Vero cell cultures

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.Peer Reviewe

Human NK Cell Subset Functions Are Differentially Affected by Adipokines

Background: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Heterotaxy Syndrome

Heterotaxy is defined as an abnormality where the internal thoraco-abdominal organs demonstrate abnormal arrangement across the left-right axis of the body. This broad term includes patients with a wide variety of very complex cardiac lesions. Patients with heterotaxy can be stratified into the subsets of asplenia syndrome and polysplenia syndrome, or the subsets of heterotaxy with isomerism of the right atrial appendages and heterotaxy with isomerism of the left atrial appendages. Treatment of patients with isomerism is determined by the nature and severity of the associated cardiac and extracardiac lesions. Most cardiac operations for patients with isomerism are palliative in nature, since normal anatomy is rarely achieved and mortality rates remain high for patients with heterotaxy syndrome. Patients with left isomerism in general have less severe cardiac malformations than those with right isomerism and, hence, more chance of biventricular repair. For almost all patients with right isomerism, and for many with left isomerism, biventricular repair will not be feasible, and all palliative protocols are then staging procedures prior to a Fontan-type repair. Recent advances in medical management, and improvements in surgical techniques have resulted in improved survival for these patients, and the surgical outcomes are comparable to those with Fontan circulation irrespective of the presence or absence of heterotaxy

Immune Status in Children Before Liver Transplantation—A Cross-Sectional Analysis Within the ChilsSFree Multicentre Cohort Study

Background: Both, markers of cellular immunity and serum cytokines have been proposed as potential biomarkers for graft rejection after liver transplantation. However, no good prognostic model is available for the prediction of acute cellular rejection. The impact of underlying disease and demographic factors on immune status before pediatric liver transplantation (pLTx) is still poorly understood. We investigated expression of immune markers before pLTx, in order to better understand the pre-transplant immune status. Improved knowledge of the impact of pre-transplant variables may enhance our understanding of immunological changes post pLTx in the future.Methods: This is a cross-sectional analysis of data from the ChilSFree study, a European multicentre cohort study investigating the longitudinal patterns of immune response before and after pLTx. Immune cell counts and soluble immune markers were measured in 155 children 1–30 days before pLTx by TruCount analysis and BioPlex assays. Results were logarithmised due to skewed distributions and then compared according to age, sex, and diagnosis using t-tests, ANOVAs, and Tukey post-hoc tests. The association between immune markers at time of pLTx and patients' age was assessed using a fractional polynomial approach. Multivariable regression models were used to assess the relative contribution of each factor.Results: Sex had no effect on immune status. We found strong evidence for age-specific differences in the immune status. The majority of immune markers decreased in a log-linear way with increasing age. T and B cells showed a sharp increase within the first months of life followed by a log-linear decline in older age groups. Several immune markers were strongly associated with underlying diagnoses. The effects of age and underlying disease remained virtually unchanged when adjusting for each other in multivariable models.Discussion: We show for the first time that age and diagnosis are major independent determinants of cellular and soluble immune marker levels in children with end-stage liver disease. These results need to be considered for future research on predictive immune monitoring after pLTx

Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design

Hepatitis A Immunity and Paediatric Liver Transplantation&mdash;A Single-Centre Analysis

Following paediatric solid organ liver transplantation, risk of infection is high, both in the short and long term. Even though an infection with hepatitis A virus (HAV) is often asymptomatic and self-limited in children, some case studies describe severe cases leading to death. Vaccinations offer simple, safe and cheap protection. However, data on vaccination rates against hepatitis A in children with liver disease are scarce. Moreover, the vaccine is only approved from the age of one year old. At the same time, up to 30% of children with liver disease are transplanted within the first year of life, so the window of opportunity for vaccination is limited. This retrospective, observational, single-centre study examines the HAV immunity in paediatric liver transplant recipients before and after the first year of transplantation. Vaccination records of 229 of 279 (82.1%) children transplanted between January 2003 and June 2021 were analysed. Of 139 eligible children aged &ge; 1 year old, only 58 (41.7%) were vaccinated at least with one HAV dose prior to transplantation. In addition, seven patients received the vaccine below one year of age. After one or two doses, 38.5% or 90.6% of 65 patients were anti-HAV-IgG positive, respectively. This percentage remained stable up to the first annual check-up. For children vaccinated only once, a shorter interval from vaccination to transplantation is a risk factor for lack of immunity. Thus, HAV immunisation should be started earlier in liver transplant candidates to improve immunity in this high-risk group

Long-Term Varicella Zoster Virus Immunity in Paediatric Liver Transplant Patients Can Be Achieved by Booster Vaccinations&mdash;A Single-Centre, Retrospective, Observational Analysis

Varicella is one of the most common vaccine-preventable infections after paediatric solid organ transplantation; thus, vaccination offers simple and cheap protection. However, children with liver disease often progress to liver transplantation (LT) before they reach the recommended vaccination age. As a live vaccine, varicella zoster virus (VZV) vaccination after transplantation is controversial; however, many case series demonstrate that vaccination may be safe and effective in paediatric liver transplant recipients. Only limited data exists describing long-term vaccination response in such immunocompromised patients. We investigated retrospectively vaccination response in paediatric patients before and after transplantation and describe long-term immunity over ten years, including the influence of booster-vaccinations. In this retrospective, single-centre study, 458 LT recipients were analysed between September 2004 and June 2021. Of these, 53 were re-transplantations. Patients with no available vaccination records and with a history of post-transplant lymphoproliferative disease, after hematopoietic stem cell transplantation and clinical chickenpox were excluded from this analysis (n = 198). In total, data on 207 children with a median annual follow-up of 6.2 years was available: 95 patients (45.9%) were unvaccinated prior to LT. Compared to healthy children, the response to vaccination, measured by seroconversion, is weaker in children with liver disease: almost 70% after one vaccination and 93% after two vaccinations. One year after transplantation, the mean titres and the number of children with protective antibody levels (VZV IgG &ge; 50 IU/L) decreased from 77.5% to 41.3%. Neither diagnosis, gender, nor age were predictors of vaccination response. Booster-vaccination was recommended for children after seroreversion using annual titre measurements and led to a significant increase in mean titre and number of protected children. Response to vaccination shows no difference from monotherapy with a calcineurin inhibitor to intensified immunosuppression by adding prednisolone or mycophenolate mofetil. Children with liver disease show weaker seroconversion rates to VZV vaccination compared to healthy children. Therefore, VZV-na&iuml;ve children should receive basic immunization with two vaccine doses as well as those vaccinated only once before transplantation. An average of 2&ndash;3 vaccine doses are required in order to achieve a long-term seroconversion and protective antibody levels in 95% of children

Hepatitis A Immunity and Paediatric Liver Transplantation—A Single-Centre Analysis

Following paediatric solid organ liver transplantation, risk of infection is high, both in the short and long term. Even though an infection with hepatitis A virus (HAV) is often asymptomatic and self-limited in children, some case studies describe severe cases leading to death. Vaccinations offer simple, safe and cheap protection. However, data on vaccination rates against hepatitis A in children with liver disease are scarce. Moreover, the vaccine is only approved from the age of one year old. At the same time, up to 30% of children with liver disease are transplanted within the first year of life, so the window of opportunity for vaccination is limited. This retrospective, observational, single-centre study examines the HAV immunity in paediatric liver transplant recipients before and after the first year of transplantation. Vaccination records of 229 of 279 (82.1%) children transplanted between January 2003 and June 2021 were analysed. Of 139 eligible children aged ≥ 1 year old, only 58 (41.7%) were vaccinated at least with one HAV dose prior to transplantation. In addition, seven patients received the vaccine below one year of age. After one or two doses, 38.5% or 90.6% of 65 patients were anti-HAV-IgG positive, respectively. This percentage remained stable up to the first annual check-up. For children vaccinated only once, a shorter interval from vaccination to transplantation is a risk factor for lack of immunity. Thus, HAV immunisation should be started earlier in liver transplant candidates to improve immunity in this high-risk group

Altered NK cell function in obese healthy humans

Background: Obesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (30 kg/m2). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme+ and IFN-γ+ NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined. Results: As opposed to similar NK cell counts, the number of CD3+CD56+ cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells. Conclusions: NK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge